Journal: EMBO Reports

Article Title: Mitotic assembly of respiratory supercomplexes ensures accurate cell division and fuels tumor growth

doi: 10.1038/s44319-026-00937-8

Figure Lengend Snippet: ( A ) The qPCR analysis of NDUFA3 mRNAs in the G1/S and mitotic HeLa cells ( n = 4 biologically independent experiments). Data with error bars are means ± SD. *** P = 0.0009 (CCNB1), * P = 0.0180 (NDUFA3). P values are determined using unpaired two-tailed Student’s t test. CCNB1 mRNA was used as a positive control. ( B ) Scatter plot of the q values of ribosome occupancy in G1/S as compared to mitotic HeLa cells(Stumpf et al, ). Red points denote the known proteins and NDUFA3 during mitosis. ( C ) Western blot analysis of cell lysates from 3xHA or NDUFA3-3xHA HeLa-Clover-ScFv cells with the indicated antibodies. ( D ) Representative immunofluorescent images of HeLa-Clover-ScFv-NDUFA3-3xHA cells stained for mitochondria (Cytochrome c, red) and DNA (DAPI, blue). Scale bar indicates 10 μm. ( E ) Western blot analysis of cell lysates from mitosis HeLa cells treated with or without MG132 or RO3306 with the indicated antibodies. ( F ) Western blot analysis of cell lysates from G1/S, and mitosis HeLa cells treated with inhibitors against CDK1 and mTOR, and immunoblotting of indicated proteins.

Article Snippet: RO3306 , Selleck , Cat# S7747.

Techniques: Analysis, Two Tailed Test, Positive Control, Western Blot, Staining

Journal: EMBO Reports

Article Title: Mitotic assembly of respiratory supercomplexes ensures accurate cell division and fuels tumor growth

doi: 10.1038/s44319-026-00937-8

Figure Lengend Snippet: ( A ) Western blot analysis of cell lysates from mitotic HeLa cells treated with inhibitors against mTOR and CDK1, and immunoblotting of indicated proteins (left). mTORi, Rapamycin; CDK1i, RO3306. CCNB1 and H3 pS10 were used as mitosis markers. Quantified data are presented ( n = 4 biologically independent experiments) (right). Data with error bars are means ± SD. (ns) P = 0.9142, * P = 0.0106, from left to right. P values are determined using one-way ANOVA. ( B ) Cartoon showing how to screen the anti-HA ScFv (Zhao et al, ) in living HeLa cells expressing NDUFA3-3xHA. ( C ) Live-cell imaging of Control, NDUFA3-3xHA or NDUFA3-3xHA with CDK1 inhibitor (RO3306) HeLa-Clover-ScFv cells. Green fluorescence indicates the expression of NDUFA3-3xHA during the cell cycle. Scale bar indicates 10 µm. ( D ) Ten representative expressions of NDUFA3-3xHA dynamics in Control, NDUFA3-3xHA or NDUFA3-3xHA with CDK1 inhibitor (RO3306) in HeLa-Clover-ScFv cells during the cell cycle. ( E ) G1/S or mitosis HeLa cells treated with or without RO3306 were collected for ribosomal protein S6-IP assay (top). The amount of NDUFA3 mRNA in ribosomal complex was determined by RT-qPCR ( n = 3 biologically independent experiments) (bottom). Data with error bars are means ± SD. *** P = 0.0002, *** P = 0.0002, from left to right. P values are determined using one-way ANOVA. ( F ) BN-PAGE immunoblots for the ETC complexes and supercomplexes of mitotic HeLa cells treated with or without RO3306 (top). Mature supercomplex forms of CI (NDUFA13, NDUFS1, NDUFV2), CIII (UQCRQ), and CIV (COX4) were quantified ( n = 3 biologically independent experiments) (bottom). Data with error bars are means ± SD. *** P = 0.0001 (NDUFA13), *** P = 0.0006 (NDUFS1), *** P = 0.0007 (NDUFV2), * P = 0.0144 (UQCRQ), ** P = 0.0023 (COX4). P values are determined using unpaired two-tailed Student’s t test. SDHA and TOM20 were used as loading controls. MPM2 was used as a mitosis marker. .

Article Snippet: RO3306 , Selleck , Cat# S7747.

Techniques: Western Blot, Analysis, Expressing, Live Cell Imaging, Control, Fluorescence, Quantitative RT-PCR, Two Tailed Test, Marker

Journal: EMBO Reports

Article Title: Mitotic assembly of respiratory supercomplexes ensures accurate cell division and fuels tumor growth

doi: 10.1038/s44319-026-00937-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: RO3306 , Selleck , Cat# S7747.

Techniques:

Journal: bioRxiv

Article Title: The TIRR–53BP1 axis controls PLK1 spatiotemporal regulation during mitosis

doi: 10.64898/2026.06.29.735294

Figure Lengend Snippet: Representative images of RPE-1 cells (control (CTL)), ΔTIRR, siCTL, siTIRR) exhibiting either ACA-positive (centric) or ACA-negative (acentric) ( a ) lagging chromosomes and ( c ) micronuclei (MN), Quantification of the percentage of cells with ( b ) lagging chromosomes and ( d ) MN (centric/acentric). ( e ) Representative images of DAPI staining showing nuclear morphology of RPE-1 control (CTL), ΔTIRR, siCTL and siTIRR cells, fixed 6 hours post-release from RO-3306-induced arrest at G2/M. ( f ) quantification of nuclear circularity and solidity for the experiment in (e). (g) Bar graphs showing the frequency of mitotic errors (lagging chromosomes, MN, alignment errors) visualized by high resolution live cell imaging of cells progressing through mitosis. (h) Bar graph depicting time from NEBD to anaphase onset determined by live cell imaging of RPE-1 cells as indicated expressing mCherry-H2B and GFP-α-Tubulin, initiated following the release from RO-3306. Data in ( b, d, g ) are mean+/-S.E from three independent experiments ( b n = 150 anaphase cells and d n =1000 cells). Data in ( f ) is represented as violin plot (n =1000 cells) from one representative experiment with three biological replicates. Bar graphs (mean+/-S.D) (h) represent mitotic cells visualized in three independent time-lapse imaging experiments (n > 35 cells each). Unpaired, two-tailed student’s t-test results as indicated. Scale bars as depicted.

Article Snippet: For synchronization in metaphase, cells were at first synchronized in G2/M by treatment with RO-3306. cells were then washed and released into media containing 20μM of proTAME (Medchem Express HY-124955) for 1 hour.

Techniques: Control, Staining, Live Cell Imaging, Expressing, Imaging, Two Tailed Test