Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: A, B (A) Equal amounts of protein extracted from WT and mutant MEFs stably expressing pLenti‐Opa1 or pLenti‐Pgs1 where indicated were separated by SDS–PAGE, immunoblotted with indicated antibodies and quantified by densitometry (B) relative to tubulin. Data represent mean ± SD of four independent experiments. C Equal amounts of protein extracted from WT MEFs treated with 20 μM CCCP (30 min) or 16 μM 4Br‐A23187 (18 h) were separated by SDS–PAGE, immunoblotted with indicated antibodies. Tubulin was used as loading control. D Representative confocal images of MEFs knocked in for mTurquoise2‐Dnm1l by Crispr/Cas9 genome editing in WT (WT Drp1KI ) and Opa1 Crispr ( Opa1 Crispr‐DrpKI ) MEFs treated with non‐targeting (NT) or Pgs1 siRNA for 72 h. DRP1 (mTurquoise2, purple), mitochondria (mitoYFP, green). Scale bar = 20 µm. E Bar graph representation of DRP1 localized to mitochondria (blue) vs cytosol (green). Data represent mean ± SD of five replicates (193–1,062 cells per cell line), One‐way ANOVA. Source data are available online for this figure.

Article Snippet: Mitochondria were stained overnight at 4°C with anti‐TOMM40 (diluted 1:1,000; ProteinTech #18409‐1‐AP) primary antibody and DRP1 with α‐DLP1 primary antibody (diluted 1:1,000, BD # 611112).

Techniques: Mutagenesis, Stable Transfection, Expressing, SDS Page, CRISPR

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: Equal amounts of protein extracted from total (T), cytosolic flow‐through (C), and mitochondrial eluate (M) from MEFs of the indicated genotypes stably expressing MitoTag (pMXs‐3XHA‐EGFP‐OMP25) obtained following mitochondrial immunocapture were separated by SDS–PAGE, immunoblotted with indicated antibody, and quantified by densitometry. Data represent mean ± SD of three independent experiments, One‐way ANOVA. Representative confocal images of MEFs of the indicated genotypes showing subcellular DRP1 distribution. Mitochondria (TOMM40, green), DRP1 labeled with anti‐DRP1 antibody (red) and nuclei (NucBlue, blue). Scale bar = 10 μm. MiD49 / 51 / Mff KO MEFs lack all 3 DRP1 receptors (MiD49, MiD51, and MFF). Bar graph representation of DRP1 localized to mitochondria (green) vs cytosol (blue). Data represent mean ± SD of three independent experiments (884–3,116 cells per cell line), unpaired t ‐test; * P < 0.05. Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected fragmentation with 5 μM carbonyl cyanide m‐chlorophenyl hydrazine (CCCP) for the indicated time points. Images were captured every hour for 18 h. Scale bar = 10 μm. Supervised ML mitochondrial morphology quantification using WT MEFs treated with 5 μM CCCP for 18 h (fragmented), untreated (normal), or treated with 10 μM CHX for 9 h (hypertubular) training sets. Data represent mean ± SD of three independent experiments (131–426 cells per cell line), One‐way ANOVA; * P < 0.05, ** P < 0.01, **** P < 0.0001, ns; not significant. FRAP fusion assay in MEFs of the indicated genotype (see Movies , , ). Scale bar = 10 μm. Quantification of mitoYFP signal intensity measured at 200 ms intervals in the photobleached area (green box) for the indicated time (seconds), represented as relative fold recovery post‐bleach. Data represent mean ± SEM of two independent experiments ( n = 18–52 cells per genotype), One‐way ANOVA. Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected hyperfusion (SiMH) with 10 μM cycloheximide (CHX) for the indicated time points. Images were captured every hour for 9 h. Mitochondrial morphology quantification of using WT MEFs treated with 5 μM CCCP for 18 h (fragmented), untreated (normal), or treated with 10 μM CHX for 9 h (hypertubular) training sets. Data represent mean ± SD of four independent experiments, (155–745 cells per cell line), One‐way ANOVA. Source data are available online for this figure.

Article Snippet: Mitochondria were stained overnight at 4°C with anti‐TOMM40 (diluted 1:1,000; ProteinTech #18409‐1‐AP) primary antibody and DRP1 with α‐DLP1 primary antibody (diluted 1:1,000, BD # 611112).

Techniques: Stable Transfection, Expressing, SDS Page, Labeling, Live Cell Imaging, Single Vesicle Fusion Assay

Journal: Frontiers in Endocrinology

Article Title: Transcriptome Analysis Reveal Candidate Genes and Pathways Responses to Lactate Dehydrogenase Inhibition (Oxamate) in Hyperglycemic Human Renal Proximal Epithelial Tubular Cells

doi: 10.3389/fendo.2022.785605

Figure Lengend Snippet: The protein expression levels of peroxisome proliferator-activated receptor-g coactivator 1α (PGC1α), caspase 3 (CASP3), B-cell lymphoma 2 apoptosis regulator (BCL2), BCL2 associated X apoptosis regulator (BAX), beclin1 (BECN1) and microtubule-associated proteins 1A/1B light chain 3 (MAP1LC3) in HK-2 cells treated with low D-glucose (LG) with different concentrations of oxamate (LGOXA-0mM, LGOXA-20mM, LGOXA-40mM and LGOXA-80mM) and high D-glucose (HG) with different concentrations of oxamate (HGOXA-0mM, HGOXA-20mM, HGOXA-40mM and HGOXA-80mM) for 24 hours. (A) Western blot gels. (B) Relative grey values. * indicates P -value < 0.05 after student’s t -test.

Article Snippet: The primary antibodies [Anti-PGC1α (ab54481), Anti-CASP3 (9665), Anti-BCL2 (ab59348), Anti-BAX (GB11690), Anti-BECN1 (bs-1353R), Anti-MAP1LC3 (14600-1-ap) and Anti-β-actin (GB12001)] and all secondary antibodies were purchased from abcom (Cambridge, MA, USA), Proteintech (Wuhan, China), BIOSS (Boston, Massachusetts, USA) and Servicebio (Wuhan, China), respectively.

Techniques: Expressing, Western Blot

Journal: Heliyon

Article Title: AP-1 inhibitor induces ferroptosis via the PI3K/AKT pathway in multiple myeloma cells

doi: 10.1016/j.heliyon.2024.e34397

Figure Lengend Snippet: The PI3K/AKT signaling pathway is involved in AP-1 inhibitor-induced ferroptosis. (A) MM cells were exposed to T-5224 (20 μМ) for 48 h. The protein levels of p -AKT(Ser473), total AKT, p-PI3K (Tyr317), and total PI3K were assessed by western blotting. (B) MM cells were exposed to T-5224 (20 μМ) for 48 h. The protein levels of Bcl2 and Bax in MM cells were assessed by western blotting. (C) MM cells were exposed to T-5224 in the presence or absence of Fer-1 (5 μM) for 48 h, after which the protein levels of p -AKT(Ser473), AKT, p-PI3K (Tyr317), and PI3K were assessed by western blotting. (D) MM cells were exposed to T-5224(20 μM) in the presence or absence of 740 Y–P (20 μM) for 48 h. The protein levels of GPX4 and SLC7A11 in MM cells were assessed by western blotting. β-actin served as the protein loading control. The results are presented as the mean ± standard deviation (SD).

Article Snippet: An anti-β-actin antibody was acquired from Proteintech.Anti-total-PI3K and anti-phospho-PI3KCA (Tyr317) antibodies were acquired from Bioss Co., Ltd. (Beijing, China).

Techniques: Western Blot, Control, Standard Deviation