Journal: bioRxiv

Article Title: Aurora kinase A/AURKA interacts with the mitochondrial ATP synthase to regulate energy metabolism and cell death

doi: 10.1101/2023.02.02.526754

Figure Lengend Snippet: ( A ) Percentage of cells with low TMRM fluorescence intensity in HEK-293 (left) and MCF7 (right) cells treated with DMSO or oligomycin for 48 h. ( B ) Percentage of HEK-293 (right) or MCF7 cells stained with the nuclear dye Hoechst 33324 to identify the phases of cell cycle, and following an incubation with DMSO or oligomycin for 48 h. ( C ) Percentage of total ATP levels in HEK-293 (left) or MCF7 (right) cells treated with DMSO or oligomycin for 48 h. ATP levels were measured using a luminescence-based assay, and were relative to the DMSO condition. ( D ) Relative HEK-293 (left) or MCF7 (right) cell number upon treatment with DMSO or oligomycin for 72 h, measured with an MTT assay. ( E ) Proportion of HEK-293 (left) or MCF7 (right) cells showing cell death features identified with PI/annexin stainings after treatment with DMSO or oligomycin for 72 h. ( F ) Cytotrack mean fluorescence intensity in HEK-293 (left) or MCF7 (right) cells treated with DMSO or oligomycin for the indicated timepoints. ( G ) Representative images and corresponding quantifications of colony-forming assays in HEK-293 (left) or MCF7 (right) cells, treated with DMSO or oligomycin for at least 2 weeks. The number of colonies in the oligomycin-treated condition is relative to that of each corresponding control. Data are means ± SD. * P <0.05,** P <0.01, *** P <0.001, and **** P <0.0001 compared to each corresponding DMSO condition (A-E, G), or to each corresponding time point in the DMSO condition (F). ns: not significant. n ≥ 3 independent experiments with at least 10,000 cells per condition quantified in A, B, E, F. n ≥ 3 independent experiments for all other panels. A. U.: arbitrary units.

Article Snippet: Mycoplasma-free HEK-293 (CRL-1573) and MCF7 (HTB-22) cells were purchased from the American Type culture collection.

Techniques: Fluorescence, Staining, Incubation, Luminescence Assay, MTT Assay

Journal: Nature Communications

Article Title: CENP-F-dependent DRP1 function regulates APC/C activity during oocyte meiosis I

doi: 10.1038/s41467-022-35461-5

Figure Lengend Snippet: a Co-IP was performed to prove the interaction between CENP-F and DRP1. Per lysate containing 1000 oocytes was incubated with anti-CENP-F and anti-DRP1, respectively. IP eluates were used for immunoblot with anti-CENP-F and anti-DRP1, respectively. The black arrow shows the band of DRP1. b The expression plasmids of PCS2 + - eGFP - Cenp-f C , PCS2 + - eGFP - Cenp-f ΔC/N or PCS2 + - eGFP - Cenp-f ΔN were co-transfected with PCS2 + - cMyc - Drp1 to HEK-293 cells for 48 h, respectively. The lysates were incubated with anti-eGFP. IP eluates were used for immunoblot with anti-eGFP and anti-cMyc, respectively. One-tenth of the input cell lysates were used for immunoblot. c Co-localisation of CENP-F and DRP1 during meiosis I. Enlarged images show representative results. White arrows indicate the measurement direction of fluorescence intensity from the inner to the outer kinetochore. The distance is shown in pixel. Scale bar, 5 μm. d , e Frequency of CENP-F and DRP1 recruited to the kinetochores during meiosis I. White arrows show the measurement direction of the fluorescence intensity. If not defined, the measurement direction starts from the left of the kinetochore to the right in the enlarged images. Fluorescence intensities of CENP-F (green line), DRP1 (red line) and CREST (magenta line) are shown in line graph. The distance shows in pixel. Scale bar, 1 μm. K, kinetochores; C, CENP-F; D, DRP1. f CENP-F and DRP1 localisation on kinetochores after CENP-F or DRP1 Trim-Away. The amplified four images in row 1, row 2 and row 3 show CENP-F and DRP1 co-localised on kinetochores, CENP-F loss reduced DRP1 localisation, and unaffected CENP-F localisation after DRP1 loss, respectively. Scale bar, 5 μm. g Fluorescence intensities of DRP1 and CENPF in ( f ) were measured. The distance is shown in pixel. Chromosomes were stained with Hoechst 33342 (blue), and kinetochores were marked with CREST (magenta in c , d , f ). n in the graphs refers to the number of kinetochores in ( e , g ). The white dotted frames in ( c , d , f ) indicate the region shown in detail. Three independent replicates were performed for ( e , g ). NEBD, nuclear envelope breakdown. Representative blots or stainings from at least three independent repeats are shown. Source data are provided as a Source Data file. See also in Supplementary Figs. – .

Article Snippet: HEK-293 cells (mycoplasma-free) were obtained from American type culture collection (ATCC, CRL-1573) and were cultured in DMEM (GIBCO) supplemented with 10% FBS (ThermoFisher) and 1% penicillin-streptomycin (GIBCO) under the humidified atmosphere of 5% CO 2 at 37 °C.

Techniques: Co-Immunoprecipitation Assay, Incubation, Western Blot, Expressing, Transfection, Fluorescence, Amplification, Staining

Journal: Cell reports

Article Title: Non-acylated Wnts can promote signaling

doi: 10.1016/j.celrep.2018.12.104

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mycoplasma-free HEK293STF (ATCC Cat# CRL-3249, RRID:CVCL_AQ26) and Expi293 (RRID:CVCL_D615) cells (human female) were cultured at 37˚C in 5% and 8% CO 2 respectively in a humidified atmosphere.

Techniques: Recombinant, Protease Inhibitor, Luciferase, SYBR Green Assay, Affinity Purification, Expressing, Software

Journal: Cell reports

Article Title: Non-acylated Wnts can promote signaling

doi: 10.1016/j.celrep.2018.12.104

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell culture Mycoplasma-free HEK293STF (ATCC Cat# CRL-3249, RRID:CVCL_AQ26) and Expi293 (RRID:CVCL_D615) cells (human female) were cultured at 37˚C in 5% and 8% CO 2 respectively in a humidified atmosphere.

Techniques: Recombinant, Protease Inhibitor, Luciferase, SYBR Green Assay, Affinity Purification, Expressing, Software