rat pheochromocytoma pc12 cells  (ATCC)

Journal: ACS Omega

Article Title: Low-Level Laser Mitigates Cisplatin-Induced Oxidative Stress and Apoptosis via AMPK-Mediated Autophagy in PC12 Cells

doi: 10.1021/acsomega.5c12039

Figure Lengend Snippet: Low-level laser (LLL) exposure improves cisplatin-induced cytotoxicity and apoptosis in PC12 cells. Cisplatin-induced reduction in cell viability (A) and increase in cytotoxicity (B) were markedly attenuated by LLL treatment at 5 J/cm 2 . (C) Apoptosis triggered by cisplatin was reduced by LLL irradiation, as determined by TUNEL assay with flow cytometry. Results are expressed as means ± SD of three independent experiments. * Indicates p < 0.05 compared with the control group.

Article Snippet: Rat pheochromocytoma PC12 cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% heat-inactivated horse serum, 5% FBS, 1% PS, and 1% l -glutamine.

Techniques: Irradiation, TUNEL Assay, Flow Cytometry, Control

Journal: ACS Omega

Article Title: Low-Level Laser Mitigates Cisplatin-Induced Oxidative Stress and Apoptosis via AMPK-Mediated Autophagy in PC12 Cells

doi: 10.1021/acsomega.5c12039

Figure Lengend Snippet: LLL enhances the cisplatin-induced autophagy in an AMPK-dependent manner. Representative Western blot images (A) and corresponding densitometric bar graphs (B, C, D) of Beclin-1, LC3-I/II, and p62 in PC12 cells exposed to cisplatin, either alone, in combination with 5 J/cm 2 LLL, or under AMPK silencing conditions, are presented. Results are expressed as means ± SD of three independent experiments. * Indicates p < 0.05 compared with the control group. # Indicates p < 0.05 compared to the cisplatin group. & Indicates p < 0.05 compared to the cisplatin + LLL group.

Article Snippet: Rat pheochromocytoma PC12 cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% heat-inactivated horse serum, 5% FBS, 1% PS, and 1% l -glutamine.

Techniques: Western Blot, Control

Journal: ACS Omega

Article Title: Low-Level Laser Mitigates Cisplatin-Induced Oxidative Stress and Apoptosis via AMPK-Mediated Autophagy in PC12 Cells

doi: 10.1021/acsomega.5c12039

Figure Lengend Snippet: LLL reduces oxidative stress and changes in mitochondrial membrane potential evoked by cisplatin through AMPK-mediated autophagy. Representative measurements of (A) malondialdehyde (MDA) and (B) mitochondrial superoxide levels in PC12 cells treated with cisplatin, and further subjected to LLL, AMPK knockdown, or rapamycin-induced autophagy. Results were expressed as means ± SD of three independent experiments. * Indicates p < 0.05 compared with the control group. # Indicates p < 0.05 compared to the cisplatin group. & Indicates p < 0.05 compared to the cisplatin + LLL group. Mitochondrial membrane potential was assessed using JC-1 staining. Fluorescence intensities in the FL-1 (green) and FL-2 (red) channels were measured by flow cytometry and quantified, with the results presented as a bar graph (C). Experimental conditions included treatment with chloroquine (an autophagy inhibitor) and MitoTEMPO (a mitochondria-targeted antioxidant), either alone or in combination with cisplatin and/or LLL.

Article Snippet: Rat pheochromocytoma PC12 cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% heat-inactivated horse serum, 5% FBS, 1% PS, and 1% l -glutamine.

Techniques: Membrane, Knockdown, Control, Staining, Fluorescence, Flow Cytometry

Journal: ACS Omega

Article Title: Low-Level Laser Mitigates Cisplatin-Induced Oxidative Stress and Apoptosis via AMPK-Mediated Autophagy in PC12 Cells

doi: 10.1021/acsomega.5c12039

Figure Lengend Snippet: LLL inhibits cisplatin-induced apoptosis through the activation of AMPK. Representative Western blot images (A) and corresponding densitometric bar graphs (B-E) of p53, Bax, Bcl-2, and cytosolic cytochrome c in PC12 cells exposed to cisplatin, either alone, in combination with 5 J/cm 2 LLL, or under AMPK silencing conditions, were presented. Results are expressed as means ± SD of three independent experiments. * Indicates p < 0.05 compared with the control group. # Indicates p < 0.05 compared to the cisplatin group. & Indicates p < 0.05 compared to the cisplatin + LLL group.

Article Snippet: Rat pheochromocytoma PC12 cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% heat-inactivated horse serum, 5% FBS, 1% PS, and 1% l -glutamine.

Techniques: Activation Assay, Western Blot, Control

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

Journal: Science Advances

Article Title: Clinically relevant stereochemistry reprograms amyloid proteome for aggregation cross-talk–conferred neuroprotection

doi: 10.1126/sciadv.aeb2729

Figure Lengend Snippet: ( A and B ) The cross-talk between L- and D-Aβ42 isomers potently suppresses neurotoxicity. While individual stereoisomers are toxic to neuronal PC12 cells (A), coincubation of all-L Aβ42 with its D-isomers in a 1:1 mixture substantially rescues cell viability, with the dD/L and dSdD/L mixtures restoring viability to near-control levels (B). The 95% confidence interval of median inhibitory concentration (IC 50 ): All-L: [17.97, 22.07]; dS: [8.591, 10.47]; dD: [21.00, 27.01]; dSdD: [38.71, 59.87]. Error bars showing the SD ( n = 5, biological replicates). (** P < 0.01 and **** P < 0.0001). ( C to E ) Quantitative proteomics reveals the molecular basis for this rescue. A four-quadrant volcano plot analysis was used to specifically isolate proteins whose expression is uniquely altered by the cross-talk effect, distinguishing them from proteins affected by the individual isomers alone. ( F and G ) Identified GO and KEGG pathways for the up-regulated (F) and down-regulated (G) proteins in STEP or cross-talk group compared with L. EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GTPases, guanosine triphosphatases. ( H ) A protein-protein interaction (PPI) network for the LD group highlights a core hub of ribosomal proteins, directly implicating the restoration of protein synthesis as a key mechanism underlying the neuroprotective effect of stereochemical cross-talk.

Article Snippet: The mouse neuroblastoma N2A cell line and rat adrenal medullary pheochromocytoma PC12 cell line were from the American Type Culture Collection (Manassas, VA).

Techniques: Control, Concentration Assay, Quantitative Proteomics, Expressing