mefs (control cells)  (StemLifeLine Inc)

Journal: Frontiers in Cell and Developmental Biology

Article Title: OPA1-Exon4b Binds to mtDNA D-Loop for Transcriptional and Metabolic Modulation, Independent of Mitochondrial Fusion

doi: 10.3389/fcell.2020.00180

Figure Lengend Snippet: OPA1-Exon4b is not required for mitochondrial fusion but ensues mitochondrial bioenergetic recovery. (A) Labeling of one subset of mitochondria by photoactivation of PAGFP in cells expressing both mtPAGFP and mtDsRed, as indicated on the top (scale bar: 10 μm). (B) Quantitation of mitochondrial fusion events, including kiss-and-run and complete fusion, per run ( n = 10 runs). (C) Δψm of WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag. The mean TMRM FI of mitochondria in WT cells was normalized to 1. The TMRM FI per mitochondrion below 0.3 denoted low (white arrow). (D) Quantitation of percentage of mitochondria with low Δψm in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag (≥20 cells for three biological replicates). (E) Quantitation of percentage of mitochondria with low Δψm in Opa1 KO cells expressing ShLuc plus Flag and Opa1 KO cells expressing OPA1-iso5-Flag plus ShLuc, Sh Mfn 1, Sh Mfn 2 or Sh Mfn 1/2 (≥20 cells for three biological replicates). ** p < 0.01, one-way ANOVA.

Article Snippet: Opa1 KO and control WT MEF cells were purchased from ATCC (Manassas, United States).

Techniques: Labeling, Expressing, Quantitation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: OPA1-Exon4b Binds to mtDNA D-Loop for Transcriptional and Metabolic Modulation, Independent of Mitochondrial Fusion

doi: 10.3389/fcell.2020.00180

Figure Lengend Snippet: OPA1 Exon-4b and Exon4 partially recover mitochondrial respiration activity. (A) OCR measurements in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag. (B) Relative ATP production, basal, and maximal respiration using OCR measurements in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag or OPA1-iso5-Flag ( n = 2, biological replicates). WT MEF cells were used as control, whose ATP production, basal, and maximal respiration were normalized to 1. (C) Cellular ATP levels in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag, with or without oligomycin treatment ( n = 3, biological replicates). * p < 0.05, ** p < 0.01, one-way ANOVA.

Article Snippet: Opa1 KO and control WT MEF cells were purchased from ATCC (Manassas, United States).

Techniques: Activity Assay, Expressing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: OPA1-Exon4b Binds to mtDNA D-Loop for Transcriptional and Metabolic Modulation, Independent of Mitochondrial Fusion

doi: 10.3389/fcell.2020.00180

Figure Lengend Snippet: OPA1-Exon4b maintains normal TFAM distribution. (A) N-SIM Images of TFAM distribution in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag. (B) Quantification of the percentage of mitochondria with diffuse TFAM in panel (A) (≥15 cells for three biological replicates, scale bar: 10 μm). (C) Anti-DNA immunofluorescence in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag (scale bar: 10 μm). (D) Quantification of mtDNA nucleoid number per cell in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag (≥20 cells for three biological replicates). * p < 0.05, ** p < 0.01, one-way ANOVA.

Article Snippet: Opa1 KO and control WT MEF cells were purchased from ATCC (Manassas, United States).

Techniques: Expressing, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: OPA1-Exon4b Binds to mtDNA D-Loop for Transcriptional and Metabolic Modulation, Independent of Mitochondrial Fusion

doi: 10.3389/fcell.2020.00180

Figure Lengend Snippet: OPA1-Exon4b modulates mtDNA transcription. (A,B) Anti-Flag ChIP was carried out using WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag. The precipitated DNA was analyzed by qPCR using primer pairs for the D-loop region (A) or Cox1 (B) . n = 3, biological replicates. (C) Anti-Flag ChIP was carried out using WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, NT-Exon4-Flag, or NT-Exon4/4b-Flag. The precipitated DNA was analyzed by qPCR using primer pairs for the D-loop region. n = 3, biological replicates. (D) Relative mRNA levels of 9 mtDNA genes in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag ( n = 3, biological replicates). (E) Relative mRNA levels of three nuclear genes encoding respiratory subunits in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag or OPA1-iso5-Flag ( n = 3, biological replicates). (F) Western blotting analysis of mtDNA-encoded Cox2 in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag or OPA1-iso5-Flag. Band densities were quantified using ImageJ, and relative band densities are shown on the bottom. n = 3, biological replicates. * p < 0.05, ** p < 0.01, one-way ANOVA.

Article Snippet: Opa1 KO and control WT MEF cells were purchased from ATCC (Manassas, United States).

Techniques: Expressing, Western Blot

Journal: Molecular cell

Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition

doi: 10.1016/j.molcel.2018.08.047

Figure Lengend Snippet:

Article Snippet: Mouse IgG2a Isotype Control from murine myeloma antibody (Pre-clear for RPA ChIP on MEF 4–3 cells) , Sigma Aldrich , M5409; RRID:AB_1163691.

Techniques: Recombinant, Purification, Transfection, DNA Labeling, Immunoprecipitation, Nucleic Acid Electrophoresis, Plasmid Preparation, Software