pc12 cells  (ATCC)

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

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

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

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

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

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