Journal: Frontiers in Cellular Neuroscience

Article Title: Mitochondrial stress disassembles nuclear architecture through proteolytic activation of PKCδ and Lamin B1 phosphorylation in neuronal cells: implications for pathogenesis of age-related neurodegenerative diseases

doi: 10.3389/fncel.2025.1549265

Figure Lengend Snippet: Mitochondrial inhibition triggers PKCδ activation and caspase-3 activity in the DAergic neuronal cell culture model. N27 cells were treated with Tebu (3 μM) for 3 h. (A) Representative immunoblots of PKCδ and phospho-PKCδ (T505) showing proteolytic cleavage-dependent activation of PKCδ and phosphorylation of PKCδ at T505 induced by Tebu; β -Actin was used as a loading control for all Western blot analyses. (B) Fluorescently stained for PKCδ (red) and phospho-PKCδ at T505 (green). (C) Caspase-3 activity assay, using the Ac-DEVD-AMC caspase-3 substrate, showing Tebu-induced caspase-3 activity in N27 cells. (D) Fluorescently stained for Caspase-3 (red) and PKCδ (green) with Hoechst dye staining the nuclei (blue). All the z -stack images in (B,D) were captured using a 63X oil immersion lens on a Leica confocal microscope. Images were processed using IMARIS 10.0 software. Scale bar, 10 μm. Data shown represent mean ± SD (* p ≤ 0.05, ** p ≤ 0.01 and **** p ≤ 0.0001) n = 3.

Article Snippet: We used the ViraPower Lentiviral gene expression system from Invitrogen (Carlsbad, CA) to generate N27 cell lines stably expressing PKCδ-WT (wild-type; WT), PKCδ-ΔNLS, and PKCδ-CRM.

Techniques: Inhibition, Activation Assay, Activity Assay, Cell Culture, Western Blot, Control, Staining, Caspase-3 Activity Assay, Microscopy, Software

Journal: Frontiers in Cellular Neuroscience

Article Title: Mitochondrial stress disassembles nuclear architecture through proteolytic activation of PKCδ and Lamin B1 phosphorylation in neuronal cells: implications for pathogenesis of age-related neurodegenerative diseases

doi: 10.3389/fncel.2025.1549265

Figure Lengend Snippet: Activated PKCδ functions as a lamin kinase, initiating Lamin B1 phosphorylation, and orchestrates lamin damage. (A) In silico and phospho site-matching analyses using the online tools NetPhos Server 2.0 and PhosphoPICK showing PKCδ site targets on Lamin B1. (B) Representative immunoblots of Lamin B1 and phospho-Lamin B1 (T575) showing that PKCδ phosphorylates Lamin B1 at T575 and induces lamin damage in Tebu-treated N27 cells; β-Actin was used as the Western blot loading control. (C) N27 cells treated with Tebu (3 μM, 3 h) were fluorescently labeled for Lamin B1 (red) and PKCδ (green) and the nuclei were stained with Hoechst blue. Scale bar, 10 μm. (D) Representative transmission electron microscopy images depicting the nuclear membrane structure of N27 cells treated with 3 μM Tebu for 3 h. The magnified inset box es point to the nuclear envelope structures of control and Tebu-treated cells. Scale bar, 1 μm. (E) Duolink proximity ligation assay reveals that PKCδ and Lamin B1 interact in Tebu-treated N27 cells but not in control cells following 3 h of pesticide exposure. Scale bar, 10 μm (* p ≤ 0.05 and ** p ≤ 0.01) n = 3.

Article Snippet: We used the ViraPower Lentiviral gene expression system from Invitrogen (Carlsbad, CA) to generate N27 cell lines stably expressing PKCδ-WT (wild-type; WT), PKCδ-ΔNLS, and PKCδ-CRM.

Techniques: In Silico, Western Blot, Control, Labeling, Staining, Transmission Assay, Electron Microscopy, Membrane, Proximity Ligation Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Mitochondrial stress disassembles nuclear architecture through proteolytic activation of PKCδ and Lamin B1 phosphorylation in neuronal cells: implications for pathogenesis of age-related neurodegenerative diseases

doi: 10.3389/fncel.2025.1549265

Figure Lengend Snippet: Stable PKCδ CRISPR/Cas9 Knockdown N27 cells and PKCδ cleavage-resistant mutant N27 cells (PKCδ-CRM) were resistant to Tebu-induced PKCδ activation and Lamin B1 phosphorylation. Representative immunoblots of (A) phospho-PKCδ (T505), (B) Lamin B1, and (C) phospho-Lamin B1 (T575) from cells treated with Tebu (3 μM, 3 h); β-Actin was used as loading control for all Western blots. (D) Immunocytochemistry analysis using confocal microscopy showing the resistance of PKCδ CRISPR/Cas9 Knockdown N27 cells to Tebu-induced PKCδ activation and Lamin B1 phosphorylation compared to control CRISPR/Cas9 N27 cells. Scale bar, 10 μm. (E,F) Representative immunoblots depicting the resistance of PKCδ-CRM N27 cells to Tebu-induced (E) PKCδ cleavage and activation and also (F) Lamin B1 phosphorylation and Lamin B1 loss (* p ≤ 0.05) n = 3–4.

Article Snippet: We used the ViraPower Lentiviral gene expression system from Invitrogen (Carlsbad, CA) to generate N27 cell lines stably expressing PKCδ-WT (wild-type; WT), PKCδ-ΔNLS, and PKCδ-CRM.

Techniques: CRISPR, Knockdown, Mutagenesis, Activation Assay, Western Blot, Control, Immunocytochemistry, Confocal Microscopy

Journal: Frontiers in Cellular Neuroscience

Article Title: Mitochondrial stress disassembles nuclear architecture through proteolytic activation of PKCδ and Lamin B1 phosphorylation in neuronal cells: implications for pathogenesis of age-related neurodegenerative diseases

doi: 10.3389/fncel.2025.1549265

Figure Lengend Snippet: Deletion of the nuclear localization sequence (NLS) of PKCδ prevented its localization with the nuclear membrane post mitochondrial dysfunctional stress and inhibited its functioning as a Lamin B1 kinase. (A) Representative immunoblots (upper panel) and densitometric analyses (lower panels) of Lamin B1 and phospho-Lamin B1 (T575) from wild-type and PKCδ-ΔNLS N27 cells treated with Tebu (3 μM, 3 h); β-Actin was used as the loading control for all Western blots. (B) Immunocytochemistry analysis using confocal microscopy showing the resistance of PKCδ-ΔNLS N27 cells to both Tebu-induced PKCδ translocation to the nuclear membrane and Lamin B1 phosphorylation compared to wild-type N27 cells. Scale bar, 10 μm. Data represent mean ± SD (* p ≤ 0.05 and **** p ≤ 0.0001) n = 2.

Article Snippet: We used the ViraPower Lentiviral gene expression system from Invitrogen (Carlsbad, CA) to generate N27 cell lines stably expressing PKCδ-WT (wild-type; WT), PKCδ-ΔNLS, and PKCδ-CRM.

Techniques: Sequencing, Membrane, Western Blot, Control, Immunocytochemistry, Confocal Microscopy, Translocation Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Mitochondrial stress disassembles nuclear architecture through proteolytic activation of PKCδ and Lamin B1 phosphorylation in neuronal cells: implications for pathogenesis of age-related neurodegenerative diseases

doi: 10.3389/fncel.2025.1549265

Figure Lengend Snippet: Site-directed mutagenesis of T575 on Lamin B1 prevented its phosphorylation-based activation and damage post-Tebu-induced oxidative stress in N27 dopaminergic neuronal cells. Representative immunoblots and densitometric analyses of (A) Lamin B1 and (B) phospho-Lamin B1 (T575) from wild-type and mutant N27 cells treated with Tebu (3 μM, 3 h); β-Actin was used as a loading control for all Western blots. Data represent mean ± SD (** p ≤ 0.01 and *** p ≤ 0.001) n = 6.

Article Snippet: We used the ViraPower Lentiviral gene expression system from Invitrogen (Carlsbad, CA) to generate N27 cell lines stably expressing PKCδ-WT (wild-type; WT), PKCδ-ΔNLS, and PKCδ-CRM.

Techniques: Mutagenesis, Activation Assay, Western Blot, Control