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ATCC pc12 cell line
Pc12 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 4437 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pc+12+cell+line/PC-12/pm41832947-71-0-3
Average 98 stars, based on 4437 article reviews

pc12 cell line - by Bioz Stars, 2026-10

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Article Title: Sequence-Independent In Situ Imaging of GlycoRNA in Living Cells and Tissues based on COMPASS
Article Snippet: Paclitaxel and 7,12-dimethylbenzanthracene (DMBA) were purchased from MedChemExpress (MCE). .. MCF-7 cell line, MCF-10A cell line, Hela cell line, HepG2 cell line, PC-12 cell line, and IMR-32 cell line were derived from ATCC. ..

Article Title: Multi-platform profiling reveals host- and cell -type-specific pseudorabies virus gene expression.
Article Snippet: .. In this study, we used the following four immortalized cell lines for the propagation of the MdBio strain of pseudorabies virus (PRV-MdBio [37]): the PK-15 porcine kidney epithelial cell line (ATCC CCL-33), the NRK rat kidney epithelial cell line (ATCC CRL-6509), the C6 cell line derived from a rat glial tumor (ATCC CCL-107), and the PC-12 cell line (ATCC CRL-1721) derived from a pheochromocytoma of the rat adrenal medulla, which has an embryonic origin from the neural crest. ..

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Article Title: Shedding light on biochemical changes in single neuron-like pheochromocytoma cells following exposure to synchrotron sourced terahertz radiation using synchrotron source Fourier transform infrared microspectroscopy
Article Snippet: Synchrotron sourced Fourier transform infrared (SS FTIR) microspectroscopy was employed to investigate the biological effects on the neuron-like pheochromocytoma (PC 12) cells after exposure to synchrotron sourced terahertz (SS THz) radiation.. Over 10 min of exposure, the PC 12 cells received a total energy of 600 J m, with a total incident power density of �1.0 W m 2 (0.10 mW cm ) at the beam extraction port (BEP) of the THz beamline at the Australian Synchrotron.. To investigate the metabolic response of PC 12 cells after synchrotron THz radiation exposure, we utilized the FTIR microscope at the Infrared Microspectroscopy IRM beamline, which offers high photon flux and diffraction-limited spatial resolution enabling the detection of functional group variations in biological molecules at a single-cell level.

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Effects of donepezil control solution, donepezil-loaded polymeric NPs, and donepezil-loaded DPPC-based liposomes treatment on ECV-304 and PC-12 cells. Cells were treated with increasing concentrations (3.125–50 µg mL −1 ) of each formulation in a time-dependent manner. All formulations maintained cell viability above 80% at concentrations up to 50 µg mL −1 in both cell lines, indicating acceptable biocompatibility. A Two-way ANOVA followed by Tukey's test was applied at each time point independently to compare the effect of different formulations across different concentrations within each cell line. **Significant with respect to the control ( p < 0.01), as shown by two-way ANOVA. The data is reported as mean ± SEM ( n = 3).

Journal: RSC Advances

Article Title: Comparative evaluation of donepezil-loaded polymeric and liposomal nanoparticles for Alzheimer's disease: biocompatibility, drug release kinetics, and cellular uptake study

doi: 10.1039/d6ra00929h

Figure Lengend Snippet: Effects of donepezil control solution, donepezil-loaded polymeric NPs, and donepezil-loaded DPPC-based liposomes treatment on ECV-304 and PC-12 cells. Cells were treated with increasing concentrations (3.125–50 µg mL −1 ) of each formulation in a time-dependent manner. All formulations maintained cell viability above 80% at concentrations up to 50 µg mL −1 in both cell lines, indicating acceptable biocompatibility. A Two-way ANOVA followed by Tukey's test was applied at each time point independently to compare the effect of different formulations across different concentrations within each cell line. **Significant with respect to the control ( p < 0.01), as shown by two-way ANOVA. The data is reported as mean ± SEM ( n = 3).

Article Snippet: The human endothelial-like cell line ECV-304 and the immortalized rat-derived neuronal-like cell line PC-12 were purchased from ATCC.

Techniques: Control, Liposomes, Formulation

In vitro cellular uptake of Rhodamine labelled DPPC-based liposomes (red) and FITC-labelled PCL/PVA nanoparticles (green) by ECV-304 and PC-12 cells after 24 hours of incubation period. Co-localization of the red fluorescence (liposomes) or green fluorescence (PCL/PVA NPs) in proximity to the blue DAPI-stained nuclei confirms the association of nanoparticles with ECV-304 and PC-12 cells, demonstrating either internalization or surface binding. Fluorescence microscopy was performed at 50× magnification; scale bar = 25 µm (consistent across all images).

Journal: RSC Advances

Article Title: Comparative evaluation of donepezil-loaded polymeric and liposomal nanoparticles for Alzheimer's disease: biocompatibility, drug release kinetics, and cellular uptake study

doi: 10.1039/d6ra00929h

Figure Lengend Snippet: In vitro cellular uptake of Rhodamine labelled DPPC-based liposomes (red) and FITC-labelled PCL/PVA nanoparticles (green) by ECV-304 and PC-12 cells after 24 hours of incubation period. Co-localization of the red fluorescence (liposomes) or green fluorescence (PCL/PVA NPs) in proximity to the blue DAPI-stained nuclei confirms the association of nanoparticles with ECV-304 and PC-12 cells, demonstrating either internalization or surface binding. Fluorescence microscopy was performed at 50× magnification; scale bar = 25 µm (consistent across all images).

Article Snippet: The human endothelial-like cell line ECV-304 and the immortalized rat-derived neuronal-like cell line PC-12 were purchased from ATCC.

Techniques: In Vitro, Liposomes, Incubation, Fluorescence, Staining, Binding Assay, Microscopy

Quantitative analysis of cellular uptake of rhodamine-labelled DPPC-based liposomes and FITC-labelled PCL/PVA NPs along with their respective free dye controls by ECV-304 and PC-12 cell lines. Both formulations showed significantly higher uptake than their respective free dye controls. PCL/PVA nanoparticles exhibited significantly higher uptake than liposomes in ECV-304 cells. Results are expressed as mean ± SD. Statistical significance was determined using a one-way ANOVA followed by Tukey's test to compare liposomes and nanoparticles uptake against their respective controls and denoted as follows: P < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

Journal: RSC Advances

Article Title: Comparative evaluation of donepezil-loaded polymeric and liposomal nanoparticles for Alzheimer's disease: biocompatibility, drug release kinetics, and cellular uptake study

doi: 10.1039/d6ra00929h

Figure Lengend Snippet: Quantitative analysis of cellular uptake of rhodamine-labelled DPPC-based liposomes and FITC-labelled PCL/PVA NPs along with their respective free dye controls by ECV-304 and PC-12 cell lines. Both formulations showed significantly higher uptake than their respective free dye controls. PCL/PVA nanoparticles exhibited significantly higher uptake than liposomes in ECV-304 cells. Results are expressed as mean ± SD. Statistical significance was determined using a one-way ANOVA followed by Tukey's test to compare liposomes and nanoparticles uptake against their respective controls and denoted as follows: P < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

Article Snippet: The human endothelial-like cell line ECV-304 and the immortalized rat-derived neuronal-like cell line PC-12 were purchased from ATCC.

Techniques: Liposomes

Metformin protects neuronal cells against Cd-induced apoptosis. Experimental timelines were tailored to specific assays: PC12 cells and primary neurons were pre-incubated with metformin (0–1.5 mM) for 24 h, followed by exposure to Cd (10 μM) for 4 h (for immunoblotting) or 24 h (for cell viability and apoptosis assays). ( A ) Cell viability was measured by MTS assay and expressed as a percentage relative to untreated controls. ( B ) Representative fluorescence micrographs illustrated apoptosis via DAPI staining (nuclear condensation/fragmentation, indicated by arrows, upper panels) and TUNEL (DNA strand breaks, green, lower panels). Scale bar: 20 μm. ( C , D ) Quantification of DAPI-positive apoptotic nuclei and TUNEL-positive cells from experiments shown in ( B ). ( E ) Whole-cell extracts were analyzed by immunoblot analysis with the specified antibodies. β-tubulin served as a loading control. Blots were representative of five independent experiments. ( F ) Densitometric analysis of cleaved caspase-3 and cleaved PARP bands normalized to β-tubulin was performed using NIH Image J software. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group.

Journal: Cells

Article Title: Metformin Alleviates Cadmium-Induced Autophagic Flux Impairment-Dependent Apoptosis by Activating AMPK in Neuronal Cells

doi: 10.3390/cells15080739

Figure Lengend Snippet: Metformin protects neuronal cells against Cd-induced apoptosis. Experimental timelines were tailored to specific assays: PC12 cells and primary neurons were pre-incubated with metformin (0–1.5 mM) for 24 h, followed by exposure to Cd (10 μM) for 4 h (for immunoblotting) or 24 h (for cell viability and apoptosis assays). ( A ) Cell viability was measured by MTS assay and expressed as a percentage relative to untreated controls. ( B ) Representative fluorescence micrographs illustrated apoptosis via DAPI staining (nuclear condensation/fragmentation, indicated by arrows, upper panels) and TUNEL (DNA strand breaks, green, lower panels). Scale bar: 20 μm. ( C , D ) Quantification of DAPI-positive apoptotic nuclei and TUNEL-positive cells from experiments shown in ( B ). ( E ) Whole-cell extracts were analyzed by immunoblot analysis with the specified antibodies. β-tubulin served as a loading control. Blots were representative of five independent experiments. ( F ) Densitometric analysis of cleaved caspase-3 and cleaved PARP bands normalized to β-tubulin was performed using NIH Image J software. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group.

Article Snippet: The rat pheochromocytoma (PC12) cell line, a widely utilized neuronal model, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Incubation, Western Blot, MTS Assay, Fluorescence, Staining, TUNEL Assay, Control, Software

Metformin mitigates Cd-induced autophagy dysregulation in neuronal cells. PC12 cells and primary neurons, either non-infected or infected with Ad-GFP-LC3, were pre-incubated with metformin (0–1.5 mM) for 24 h, followed by exposure to Cd (10 μM) for 4 h (for Immunoblotting) or 12 h (for MDC staining and GFP-LC3 puncta analysis). ( A , B ) Following MDC labeling to visualize autophagic vacuoles, representative fluorescence micrographs ( A ) and quantitative analysis of MDC-positive vacuole fluorescence intensity ( B ) were presented. Scale bar: 20 μm. ( C , D ) Autophagosome formation was monitored via GFP-LC3 redistribution; representative images ( C ) and quantification of GFP-LC3 puncta per cell ( D ) demonstrated autophagic flux alterations. Scale bar: 2 μm. ( E ) Immunoblot analysis of autophagy-related markers was performed on whole-cell lysates using antibodies against ATG5, LC3, and p62, with β-tubulin serving as the loading control. Blots were representative of five independent experiments. ( F ) Densitometric quantification of ATG5, LC3-II, and p62 levels normalized to β-tubulin was performed using NIH Image J software. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group.

Journal: Cells

Article Title: Metformin Alleviates Cadmium-Induced Autophagic Flux Impairment-Dependent Apoptosis by Activating AMPK in Neuronal Cells

doi: 10.3390/cells15080739

Figure Lengend Snippet: Metformin mitigates Cd-induced autophagy dysregulation in neuronal cells. PC12 cells and primary neurons, either non-infected or infected with Ad-GFP-LC3, were pre-incubated with metformin (0–1.5 mM) for 24 h, followed by exposure to Cd (10 μM) for 4 h (for Immunoblotting) or 12 h (for MDC staining and GFP-LC3 puncta analysis). ( A , B ) Following MDC labeling to visualize autophagic vacuoles, representative fluorescence micrographs ( A ) and quantitative analysis of MDC-positive vacuole fluorescence intensity ( B ) were presented. Scale bar: 20 μm. ( C , D ) Autophagosome formation was monitored via GFP-LC3 redistribution; representative images ( C ) and quantification of GFP-LC3 puncta per cell ( D ) demonstrated autophagic flux alterations. Scale bar: 2 μm. ( E ) Immunoblot analysis of autophagy-related markers was performed on whole-cell lysates using antibodies against ATG5, LC3, and p62, with β-tubulin serving as the loading control. Blots were representative of five independent experiments. ( F ) Densitometric quantification of ATG5, LC3-II, and p62 levels normalized to β-tubulin was performed using NIH Image J software. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group.

Article Snippet: The rat pheochromocytoma (PC12) cell line, a widely utilized neuronal model, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Infection, Incubation, Western Blot, Staining, Labeling, Fluorescence, Control, Software

Downregulation of ATG5 augments metformin’s suppression of Cd-induced autophagosome formation and apoptosis in neuronal cells. PC12 cells were transduced with lentiviral particles encoding shRNA targeting ATG5 or a non-targeting control (GFP shRNA). Following transduction, cells were optionally infected with Ad-GFP-LC3 for subsequent autophagosome visualization, then subjected to metformin pretreatment (1 mM) for 24 h and Cd (10 μM) challenge for 4 h (for immunoblotting), 12 h (for GFP-LC3 puncta analysis), or 24 h (for TUNEL staining). ( A ) Whole-cell extracts were analyzed by immunoblot analysis with the specified antibodies. β-tubulin served as a loading control. Blots were representative of five independent experiments. ( B ) Densitometric quantification of ATG5, LC3-II, and cleaved caspase-3 levels normalized to β-tubulin was performed using NIH Image J software. ( C ) Representative fluorescence micrographs depicted the distribution of GFP-LC3 puncta (green) in the cells. Scale bar: 20 μm. ( D ) Quantitative analysis of autophagosome abundance, expressed as the number of GFP-LC3 puncta per cell, reflected the net effect of ATG5 knockdown on autophagic vacuole accumulation. ( E ) Quantification of apoptotic cells was shown via TUNEL staining, visualizing nuclear DNA strand breaks. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group. c p < 0.05 ATG5 shRNA group versus GFP shRNA group.

Journal: Cells

Article Title: Metformin Alleviates Cadmium-Induced Autophagic Flux Impairment-Dependent Apoptosis by Activating AMPK in Neuronal Cells

doi: 10.3390/cells15080739

Figure Lengend Snippet: Downregulation of ATG5 augments metformin’s suppression of Cd-induced autophagosome formation and apoptosis in neuronal cells. PC12 cells were transduced with lentiviral particles encoding shRNA targeting ATG5 or a non-targeting control (GFP shRNA). Following transduction, cells were optionally infected with Ad-GFP-LC3 for subsequent autophagosome visualization, then subjected to metformin pretreatment (1 mM) for 24 h and Cd (10 μM) challenge for 4 h (for immunoblotting), 12 h (for GFP-LC3 puncta analysis), or 24 h (for TUNEL staining). ( A ) Whole-cell extracts were analyzed by immunoblot analysis with the specified antibodies. β-tubulin served as a loading control. Blots were representative of five independent experiments. ( B ) Densitometric quantification of ATG5, LC3-II, and cleaved caspase-3 levels normalized to β-tubulin was performed using NIH Image J software. ( C ) Representative fluorescence micrographs depicted the distribution of GFP-LC3 puncta (green) in the cells. Scale bar: 20 μm. ( D ) Quantitative analysis of autophagosome abundance, expressed as the number of GFP-LC3 puncta per cell, reflected the net effect of ATG5 knockdown on autophagic vacuole accumulation. ( E ) Quantification of apoptotic cells was shown via TUNEL staining, visualizing nuclear DNA strand breaks. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group. c p < 0.05 ATG5 shRNA group versus GFP shRNA group.

Article Snippet: The rat pheochromocytoma (PC12) cell line, a widely utilized neuronal model, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Transduction, shRNA, Control, Infection, Western Blot, TUNEL Assay, Staining, Software, Fluorescence, Knockdown

Metformin alleviates Cd-triggered autophagic flux impairment and attenuates apoptosis resulting from autophagosome accumulation in neuronal cells. PC12 cells and primary neurons, either infected with Ad-GFP-LC3 or left uninfected, were pre-incubated with/without CQ (25 μM) for 1 h, followed by metformin (1 mM) pretreatment for 24 h, and subsequently exposed in the presence or absence of Cd (10 μM) for specified durations—4 h for immunoblot analysis, 12 h for evaluation of GFP-LC3 puncta, or 24 h for detection of DNA fragmentation via TUNEL staining. ( A ) Immunoblotting of whole-cell extracts using antibodies against ATG5, LC3, p62, cleaved caspase-3, and β-tubulin (loading control). Blots were representative of five independent experiments. ( B ) Densitometric quantification of ATG5, LC3-II, p62, and cleaved caspase-3 levels normalized to β-tubulin was performed using NIH Image J software. ( C ) Quantitative analysis of autophagosome abundance was expressed as the number of GFP-LC3 puncta per cell. ( D ) Quantification of apoptotic cells was shown via TUNEL staining, visualizing nuclear DNA strand breaks. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group. c p < 0.05 compared to Cd/Metformin or Cd/CQ co-treatment group.

Journal: Cells

Article Title: Metformin Alleviates Cadmium-Induced Autophagic Flux Impairment-Dependent Apoptosis by Activating AMPK in Neuronal Cells

doi: 10.3390/cells15080739

Figure Lengend Snippet: Metformin alleviates Cd-triggered autophagic flux impairment and attenuates apoptosis resulting from autophagosome accumulation in neuronal cells. PC12 cells and primary neurons, either infected with Ad-GFP-LC3 or left uninfected, were pre-incubated with/without CQ (25 μM) for 1 h, followed by metformin (1 mM) pretreatment for 24 h, and subsequently exposed in the presence or absence of Cd (10 μM) for specified durations—4 h for immunoblot analysis, 12 h for evaluation of GFP-LC3 puncta, or 24 h for detection of DNA fragmentation via TUNEL staining. ( A ) Immunoblotting of whole-cell extracts using antibodies against ATG5, LC3, p62, cleaved caspase-3, and β-tubulin (loading control). Blots were representative of five independent experiments. ( B ) Densitometric quantification of ATG5, LC3-II, p62, and cleaved caspase-3 levels normalized to β-tubulin was performed using NIH Image J software. ( C ) Quantitative analysis of autophagosome abundance was expressed as the number of GFP-LC3 puncta per cell. ( D ) Quantification of apoptotic cells was shown via TUNEL staining, visualizing nuclear DNA strand breaks. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group. c p < 0.05 compared to Cd/Metformin or Cd/CQ co-treatment group.

Article Snippet: The rat pheochromocytoma (PC12) cell line, a widely utilized neuronal model, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Infection, Incubation, Western Blot, TUNEL Assay, Staining, Control, Software

Metformin sustains autophagic flux and attenuates Cd-elicited apoptosis through reactivation of the AMPK signaling axis. PC12 cells and primary neurons, with/without Ad-GFP-LC3 infection, were pretreated with metformin (0–1.5 mM) for 24 h, or pretreated with AICAR (2 mM, 1 h) followed by metformin (1 mM), then exposed to Cd (10 μM) for 4 h (for Immunoblotting), 12 h (for GFP-LC3 puncta analysis) or 24 h (for TUNEL staining). ( A , C ) Whole-cell extracts were analyzed by immunoblot analysis with the specified antibodies. β-tubulin served as a loading control. Blots were representative of five independent experiments. ( B , D ) Densitometric quantification of p-AMPKα, p-ACC, ATG5, LC3-II, p62, and cleaved caspase-3 levels normalized to β-tubulin was performed using NIH Image J software. ( E ) Quantitative analysis of autophagosome abundance was expressed as the number of GFP-LC3 puncta per cell. ( F ) Quantification of apoptotic cells was shown via TUNEL staining, visualizing nuclear DNA strand breaks. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group. c p < 0.05 compared to Cd/Metformin or Cd/AICAR co-treatment group.

Journal: Cells

Article Title: Metformin Alleviates Cadmium-Induced Autophagic Flux Impairment-Dependent Apoptosis by Activating AMPK in Neuronal Cells

doi: 10.3390/cells15080739

Figure Lengend Snippet: Metformin sustains autophagic flux and attenuates Cd-elicited apoptosis through reactivation of the AMPK signaling axis. PC12 cells and primary neurons, with/without Ad-GFP-LC3 infection, were pretreated with metformin (0–1.5 mM) for 24 h, or pretreated with AICAR (2 mM, 1 h) followed by metformin (1 mM), then exposed to Cd (10 μM) for 4 h (for Immunoblotting), 12 h (for GFP-LC3 puncta analysis) or 24 h (for TUNEL staining). ( A , C ) Whole-cell extracts were analyzed by immunoblot analysis with the specified antibodies. β-tubulin served as a loading control. Blots were representative of five independent experiments. ( B , D ) Densitometric quantification of p-AMPKα, p-ACC, ATG5, LC3-II, p62, and cleaved caspase-3 levels normalized to β-tubulin was performed using NIH Image J software. ( E ) Quantitative analysis of autophagosome abundance was expressed as the number of GFP-LC3 puncta per cell. ( F ) Quantification of apoptotic cells was shown via TUNEL staining, visualizing nuclear DNA strand breaks. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group. c p < 0.05 compared to Cd/Metformin or Cd/AICAR co-treatment group.

Article Snippet: The rat pheochromocytoma (PC12) cell line, a widely utilized neuronal model, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Infection, Western Blot, TUNEL Assay, Staining, Control, Software

Constitutive AMPKα activation strengthens metformin’s alleviation of Cd-induced autophagic flux impairment-dependent apoptosis in neuronal cells. PC12 cells were engineered to express a constitutively active AMPKα mutant (Ad-AMPKα-ca) or GFP control (Ad-GFP), with/without subsequent Ad-GFP-LC3 or Ad-mCherry-GFP-LC3 infection for autophagic flux visualization. Cells were pretreated with/without metformin (1 mM) for 24 h, followed by exposure to Cd (10 μM) for 4 h (for immunoblotting), 12 h (for GFP-LC3 puncta and mCherry-GFP-LC3 tandem reporter assays), or 24 h (for TUNEL staining). ( A ) Whole-cell extracts were analyzed by immunoblot analysis with the specified antibodies. β-tubulin served as a loading control. Blots were representative of five independent experiments. ( B ) Densitometric quantification of p-AMPKα, p-ACC, ATG5, LC3-II, p62, and cleaved caspase-3 levels normalized to β-tubulin was performed using NIH Image J software. ( C ) Representative fluorescence micrographs depicted the distribution of GFP-LC3 puncta (green) in the cells. Scale bar: 20 μm. ( D ) Quantitative analysis of autophagosome abundance was expressed as the number of GFP-LC3 puncta per cell. ( E ) Quantification of apoptotic cells was shown via TUNEL staining, visualizing nuclear DNA strand breaks. ( F ) Representative images of tandem fluorescent mCherry-GFP-LC3 reporters: GFP signal (green) quenched in acidic lysosomal compartments, while mCherry signal (red) remains stable. Co-localized GFP + /mCherry + -LC3 (yellow in merge) puncta indicate autophagosomes that have not undergone lysosomal fusion. Scale bar: 2 μm. ( G ) Quantitative analysis of autophagic flux status was presented as the mean number of yellow (GFP + /mCherry + -LC3) puncta per cell. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group. c p < 0.05 Ad-AMPKα-ca group versus Ad-GFP group.

Journal: Cells

Article Title: Metformin Alleviates Cadmium-Induced Autophagic Flux Impairment-Dependent Apoptosis by Activating AMPK in Neuronal Cells

doi: 10.3390/cells15080739

Figure Lengend Snippet: Constitutive AMPKα activation strengthens metformin’s alleviation of Cd-induced autophagic flux impairment-dependent apoptosis in neuronal cells. PC12 cells were engineered to express a constitutively active AMPKα mutant (Ad-AMPKα-ca) or GFP control (Ad-GFP), with/without subsequent Ad-GFP-LC3 or Ad-mCherry-GFP-LC3 infection for autophagic flux visualization. Cells were pretreated with/without metformin (1 mM) for 24 h, followed by exposure to Cd (10 μM) for 4 h (for immunoblotting), 12 h (for GFP-LC3 puncta and mCherry-GFP-LC3 tandem reporter assays), or 24 h (for TUNEL staining). ( A ) Whole-cell extracts were analyzed by immunoblot analysis with the specified antibodies. β-tubulin served as a loading control. Blots were representative of five independent experiments. ( B ) Densitometric quantification of p-AMPKα, p-ACC, ATG5, LC3-II, p62, and cleaved caspase-3 levels normalized to β-tubulin was performed using NIH Image J software. ( C ) Representative fluorescence micrographs depicted the distribution of GFP-LC3 puncta (green) in the cells. Scale bar: 20 μm. ( D ) Quantitative analysis of autophagosome abundance was expressed as the number of GFP-LC3 puncta per cell. ( E ) Quantification of apoptotic cells was shown via TUNEL staining, visualizing nuclear DNA strand breaks. ( F ) Representative images of tandem fluorescent mCherry-GFP-LC3 reporters: GFP signal (green) quenched in acidic lysosomal compartments, while mCherry signal (red) remains stable. Co-localized GFP + /mCherry + -LC3 (yellow in merge) puncta indicate autophagosomes that have not undergone lysosomal fusion. Scale bar: 2 μm. ( G ) Quantitative analysis of autophagic flux status was presented as the mean number of yellow (GFP + /mCherry + -LC3) puncta per cell. Data are expressed as mean ± SEM ( n = 5). a p < 0.05 compared to control group; b p < 0.05 compared to 10 μM Cd alone group. c p < 0.05 Ad-AMPKα-ca group versus Ad-GFP group.

Article Snippet: The rat pheochromocytoma (PC12) cell line, a widely utilized neuronal model, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activation Assay, Mutagenesis, Control, Infection, Western Blot, TUNEL Assay, Staining, Software, Fluorescence

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