Journal: Developmental cell

Article Title: Mitotic Cortical Waves Predict Future Division Sites by Encoding Positional and Size Information.

doi: 10.1016/j.devcel.2017.10.023

Figure Lengend Snippet: Figure 2. Cells with Metaphase Waves Experience Longer Metaphase and Require SAC Signals (A) Intensity plots from representative cells with (top two traces) or without (bottom trace) metaphase waves. The arrowhead indicates the beginning of each mitotic stage. NEBD, nuclear envelop break down. Scale bar represents 5 min. (B) The ratio of cortical wave amplitude (mitosis/interphase) in cells without metaphase waves (n = 56 cells) and cells with metaphase waves (n = 53 cells). The red dashed line indicates a threshold value of 0.5. The data are presented as means ± SD. (C) Normalized amplitudes from cells with mitotic wave (n = 54 cells) and without mitotic wave (n = 56 cells). Data are shown as means ±SD. (D) Time duration of each mitotic stage of cells with metaphase wave (n = 24 cells) and cells without target wave (n = 18 cells). The data are presented as means ± SD. (E) Quantification of the percentage of cells displaying metaphase waves. Wild-type (WT, n = 149 cells), ZM447439 (Aurora B Kinase inhibitor, n = 39 cells), and MPS1 knockdown (n = 41 cells). (F) Kymograph of cells stably expressing EGFP-CBD treated with 10 mM Mps1-IN-1. Intensity plot is also shown. Horizontal scale bar represents 1 min, vertical scale bar represents 10 mm. t test: **p % 0.01; ns, not significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, Peptides, and Recombinant Proteins Thymidine Sigma-Aldrich Cat#: T1895 Cytochalasin D Sigma-Aldrich Cat#: C8273 ZM447439 Millipore Cat#:189410 Mps1-IN-1 Tocris Cat#: 5142 Monastrol Sigma-Aldrich Cat#: M8515 Nocodazole Sigma-Aldrich Cat#: M1404 Wiskostatin Sigma-Aldrich Cat#: W2270 Manganese chloride tetrahydrate Sigma-Aldrich Cat#: M3634 RO3306 Sigma-Aldrich Cat#: SML0569 Aphidicolin Sigma-Aldrich Cat#: A0781 SiR-Tubulin Cytoskeleton Cat#: SC002 Experimental Models: Cell Lines RBL-2H3 cell ATCC Cat#: CRL-2256; RRID:CVCL_0591 Oligonucleotides SMARTpool siRNA for MPS1: 5’-CGGAAGUAAGUCAUCGAAA-3’ Thermo Fisher Cat#: L-080276-02-0005 SMARTpool siRNA for MPS1: 5’-GGAUCAGAAUACCGAGAUU-3’ Thermo Fisher Cat#: L-080276-02-0005 SMARTpool siRNA for MPS1: 5’-CUAUAMGACCUACGGGAAA-3’ Thermo Fisher Cat#: L-080276-02-0005 SMARTpool siRNA for MPS1: 5’-CCAAAACACUAAACGGAUU-3’ Thermo Fisher Cat#: L-080276-02-0005 Recombinant DNA EGFP-FBP17 Wu et al., 2013 N/A mCherry-SHIP1 Xiong et al., 2016 N/A EGFP-CBD Wu et al., 2013 N/A mRFP-CBD Addgene Cat#: 26733 mTagRFP-H2B Addgene Cat #: 58091 EGFP-Anillin Gift from W. Huttner N/A mRuby-LifeAct Gift from R. Wedlich-Soldner N/A Rap1-Q63E Addgene Cat #: 32698 Rap1-S17A Gift from B. Baum N/A Software and Algorithms Fiji (image J) NIH http://fiji.sc/ Prism 6 Graphpad N/A

Techniques: Knockdown, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: MtDNA heteroplasmy controls tumor immune reprogramming through mitochondrial translation

doi: 10.64898/2026.01.31.693596

Figure Lengend Snippet: a Representative tumor images (top left), including PyMT- TrnE Low, Mid, and High-mutant groups (mtDNA mutation loads indicated on the graph), and tumor weight quantification (right) at 20 weeks. n = 3, 5, and 3 mice for PyMT- TrnE Low, Mid, and High groups, respectively. Schematic illustration (bottom left) showing the workflow for tumor dissociation and live cell sorting for scRNA-seq. b Gene expression from scRNA-seq experiments characterizing lineage-defining markers across cell clusters in PyMT- TrnE 20w tumor models. c Integrated Uniform Manifold Approximation and Projection (UMAP) plot and cell annotation of scRNA-seq data from tumors of PyMT- TrnE Low, Mid, and High groups at 20 weeks. d Volcano plots of tumor cell clusters from scRNA-seq data at 20 weeks showing top differentially expressed genes between groups, with OXPHOS subunits labeled in purple, MRPL/MRPS in blue, and RPL/RPS in pink. Comparisons include PyMT- TrnE Mid vs Low (left), PyMT- TrnE Mid vs High (center), and PyMT- TrnE High vs Low (right). Genes with FDR-adjusted two-tailed t-test P < 0.05 were considered statistically significant. e Bubble plot showing pathway enrichment in tumor cell clusters from 20w PyMT- TrnE Low, Mid, and High tumors, with comparisons of Mid vs Low (top left), Mid vs High (top right), and High vs Low (bottom), where bubble size represents gene count and color intensity indicates adjusted p-value (padj). f, g Representative immunofluorescence staining (f) and quantification (g) of high RPL28 expression in tumor cells (Ep-CAM + ) from PyMT- TrnE Low, Mid, and High mice at 20 weeks of age (n = 18 fields from 3 mice per group). Scale bars, 100 μm. h, i Immunoblots (h) showing expression levels of ribosomal protein, mitochondrial OXPHOS proteins, mitochondrial transport and folding proteins, and mitochondrial ribosomal proteins in PyMT TrnE tumor cells at the early 20-week stage. Bar graphs (i) showing quantification of RPS27, Complex I-V, HSP60, TOMM40, TOMM20, MRPL28, and MRPS30 expression across PyMT TrnE Low, Mid, and High-mutant groups at 20 weeks. β-Actin serves as a loading control. n = 3 biological replicates. Data are as mean ± s.e.m. One-way ANOVA with Tukey’s multiple comparison test ( a, g, i ). Schematic in a (bottom left) were created in BioRender. NS, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Membranes were blocked with TBST buffer containing 5% non-fat milk for 1 h and incubated with the primary antibody in primary antibody diluent (10X, Beyotime) overnight at 4 °C: MFN2 (ABclonal A19678), DRP1 (Abcam ab184248), HSP60 (Abcam ab190828), VDAC1 (Merck MABN504), TFAM (Abcam ab131607), OXPHOS (Abcam ab110413), GAPDH (Proteintech 81640-5-RR), PLOG (Abcam ab128899), MRPS30 (Proteintech 18441-1-AP), MRPL28 (Proteintech 68467-1-Ig), RPS27 (Proteintech 15355-1-AP), LARS2 (Affinity DF13122), TOMM40 (ThermoFisher PA5-110507), TOMM20 (Cell Signaling Technology D8T4N) and β-actin (ABclonal AC026).

Techniques: Mutagenesis, FACS, Gene Expression, Labeling, Two Tailed Test, Immunofluorescence, Staining, Expressing, Western Blot, Control, Comparison