Journal: International journal of molecular sciences

Article Title: The Mitochondrial PHB2/OMA1/DELE1 Pathway Cooperates with Endoplasmic Reticulum Stress to Facilitate the Response to Chemotherapeutics in Ovarian Cancer.

doi: 10.3390/ijms23031320

Figure Lengend Snippet: Figure 2. FCCP combined with cisplatin activated OMA1 and induced optic atrophy 1 (OPA1) cleavage and mitochondrial fragmentation. A2780 cells were treated with FCCP (2.5 µM) and/or cisplatin (1 µg/mL) for 6 and 12 h. ID8 cells were treated with FCCP (5 µM) and/or cisplatin (4 µg/mL) for 6 and 12 h. The expression of PARL, OMA1, OPA1, and PGAM5 was measured by Western blotting (a–d). A2780 cells were treated with FCCP (2.5 µM) and/or cisplatin (1 µg/mL) for 12 h, stained with Hoechst 33342 and MitoTracker Red and observed under a fluorescence microscope (scale bar, 30 µm) (e).

Article Snippet: Mitochondrial Isolation was performed using the Mitochondrial Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA) following the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Staining, Microscopy

Journal: International journal of molecular sciences

Article Title: The Mitochondrial PHB2/OMA1/DELE1 Pathway Cooperates with Endoplasmic Reticulum Stress to Facilitate the Response to Chemotherapeutics in Ovarian Cancer.

doi: 10.3390/ijms23031320

Figure Lengend Snippet: Figure 3. FCCP combined with cisplatin induced mitochondrial inner membrane (MIM) cristae remodeling and cytochrome c release. A2780 cells were treated with FCCP (2.5 µM) and/or cisplatin (1 µg/mL) for 12 h and observed under transmission electron microscopy. Red arrowheads indicated intact mitochondrial membranes. Green arrowheads indicated damaged mitochondrial membranes (scale bar, 0.5 µm) (a). A2780 cells were treated with FCCP (2.5 µM) and/or cisplatin (1 µg/mL) for 12 h. ID8 cells were treated with FCCP (5 µM) and/or cisplatin (4 µg/mL) for 12 h. The expression of cytochrome c in cytoplasm was measured by Western blotting (b,c). Mitochondrial membrane potentials were measured by JC-1 (d,e).

Article Snippet: Mitochondrial Isolation was performed using the Mitochondrial Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA) following the manufacturer’s protocol.

Techniques: Membrane, Transmission Assay, Electron Microscopy, Expressing, Western Blot

Journal: International journal of molecular sciences

Article Title: The Mitochondrial PHB2/OMA1/DELE1 Pathway Cooperates with Endoplasmic Reticulum Stress to Facilitate the Response to Chemotherapeutics in Ovarian Cancer.

doi: 10.3390/ijms23031320

Figure Lengend Snippet: Figure 5. Knockdown of OMA1 partially reversed apoptosis by reducing the mitochondrial stress and the EIF2S1/ATF4 pathway. A2780 cells were transfected with OMA1-shRNA plasmids and Scr- shRNA for 24 h. After treatment with FCCP (2.5 µM) and/or cisplatin (1 µg/mL) for 24 h, apoptosis rates (a,b) and cell viability (c) were measured. Data are presented as mean ± SD, n = 3. ** p < 0.01 vs. FCCP + cisplatin + Src-shRNA. After treatment for 12 h, the expression of OMA1 and OPA1 (d) in the whole cell and cytochrome c in the cytoplasm (h) was evaluated. After treatment for 12 h, A2780 cells were stained with Hoechst 33342 and MitoTracker Red and observed under a fluorescence microscope (scale bar, 30 µm) (e). The wild-type A2780 cells and the OMA1 stable knockdown A2780 cells were transfected with DELE1-HA for 24 h, then treated with FCCP (2.5 µM) and/or cisplatin (1 µg/mL) for 6 h. Expression of DELE1 was evaluated (f). A2780 cells were transfected with OMA1-shRNA plasmids and Scr-shRNA for 24 h. After treatment with FCCP (2.5 µM) and/or cisplatin (1 µg/mL) for 6 h, the expression of p-EIF2S1, EIF2S1, ATF4, ATF5, and DDIT3 was evaluated (g).

Article Snippet: Mitochondrial Isolation was performed using the Mitochondrial Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA) following the manufacturer’s protocol.

Techniques: Knockdown, Transfection, shRNA, Expressing, Staining, Microscopy

Journal: International journal of molecular sciences

Article Title: The Mitochondrial PHB2/OMA1/DELE1 Pathway Cooperates with Endoplasmic Reticulum Stress to Facilitate the Response to Chemotherapeutics in Ovarian Cancer.

doi: 10.3390/ijms23031320

Figure Lengend Snippet: Figure 8. The mitochondrial PHB2/OMA1/DELE1 pathway cooperates with endoplasmic reticulum stress to induce ovarian cancer cell death. Destruction of the prohibitin 2 (PHB2)/stomatin-like protein 2 (STOML2) complex releases OMA1 protease activity. Activated OMA1 cleaves optic atrophy 1 (OPA1) and DELE1, resulting in mitochondrial cristae remodeling and DELE1’s cytoplasmic interac- tion with EIF2AK1. Activated EIF2AK1 cooperates with EIF2AK3 to induce EIF2S1 phosphorylation, promoting the transfer of ATF4 into the nucleus and the upregulation of DDIT3 and ATF5. Then, pro-apoptotic proteins, BBC3, BCL2L11 and PMAIP1, are upregulated and anti-apoptotic protein MCL1 is downregulated, triggering accumulation of BAX and BAK1 on the mitochondrial outer membrane (MOM) and the rupture of the MOM. Under the coordination of mitochondrial inner and outer membranes, cytochrome c is released, and the cell dies. Green arrows indicate positive regulation; red arrow indicates negative regulation.

Article Snippet: Mitochondrial Isolation was performed using the Mitochondrial Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA) following the manufacturer’s protocol.

Techniques: Activity Assay, Phospho-proteomics, Membrane

Journal: JCI insight

Article Title: A modifier screen identifies DNAJB6 as a cardiomyopathy susceptibility gene.

doi: 10.1172/jci.insight.88797

Figure Lengend Snippet: Figure 2. The GBT0411 mutant was tagged to the dnajb6b(L) isoform that predominantly expresses in the heart. (A) Northern blot analysis of dnajb6b(S) and dnajb6b(L) isoforms in indicated 3-month-old adult fish tissues demonstrated a predominant expression of dnajb6b(L) in the fish heart and skeletal muscle. (B) Western blot analysis (left) and quantification (right) of Dnajb6(S) and Dnajb6(L) isoforms expression in 1-year-old WT C57BL/6 mouse tissues indicated a predominant expression of Dnajb6(L) in the mouse heart. Values are presented relative to heart expression. Data represent mean ±SEM. (C) Western blot analysis (left) and quantification (right) of DNAJB6(L) and DNAJB6(S) protein expression in human heart and skeletal muscle lysates. Values are presented relative to heart expression. Data represent mean ±SEM. (D) Kaplan-Meier survival curves of indicated fish injected with a single bolus of 20 μg/gbm doxorubicin (DOX). Cardiomyocyte-specific overexpression of zebrafish dnajb6b(L) rescued DOX-induced fish death in the GBT0411 heterozygous mutant. *P < 0.05, log-rank test. Comparison of Tg(cmlc2:dnajb6b(L)-EGFP); GBT0411+/– vs. GBT0411+/–.

Article Snippet: Human heart and skeletal muscle tissue lysates were purchased from Novus Biologicals LLC.

Techniques: Mutagenesis, Northern Blot, Expressing, Western Blot, Injection, Over Expression, Comparison