Journal: The Journal of Cell Biology

Article Title: Non-cell autonomous regulation of cell–cell signaling and differentiation by mitochondrial ROS

doi: 10.1083/jcb.202401084

Figure Lengend Snippet: Reagents

Article Snippet: Rabbit polyclonal anti-phospho-Histone H3 (Ser10) , Cell Signaling Technology , 9701S.

Techniques: Virus, Recombinant, Membrane, Protein Extraction, Over Expression, Software

Journal: eLife

Article Title: Mammalian cell growth dynamics in mitosis

doi: 10.7554/eLife.44700

Figure Lengend Snippet: ( a ) Top, schematic of the protocol for quantifying mitotic protein synthesis rates using O-propargyl-puromycin (OPP). G2 synchronization was achieved by double thymidine block followed by RO-3306 mediated G2 arrest. Bottom, representative FACS scatter plots indicating L2110 cell cycle synchrony (n = 3 independent experiments). Phospho-Histone H3 (Ser10) was used as a mitotic marker. ( b ) Ratio of protein synthesis rate (blue) between mitotic and G2 L1210 cells after release from G2 arrest. Light green area displays the typical protein synthesis ratio between mitotic and G2 cells in the absence of cell cycle synchronization. The relative portion of mitotic cells is shown in orange. Each data point represents an individual replicate. (n = 3 separate cultures for each timepoint). Time of G2 release and the typical time to reach metaphase-to-anaphase transition are indicated with dashed vertical lines. ( c ) Representative FACS scatter plot indicating the separation of early (prophase) and late (metaphase to telophase) mitotic L1210 cells using Cyclin A antibody staining. ( d ) Protein synthesis rate of G2, early mitotic and late mitotic L1210 cells. (n = 6 separate cultures). Early and late mitotic cells were separated as shown in ( g ). p-Values obtained using ANOVA followed by Tukey’s posthoc test.

Article Snippet: Antibody , Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit monoclonal Ab (Alexa Fluor 488 Conjugate) , Cell Signaling Technology , Cat#3465; RRID: AB_10695860 , Dilution 1/100 in PBS supplemented with 5% BSA.

Techniques: Blocking Assay, Marker, Staining

Journal: eLife

Article Title: Mammalian cell growth dynamics in mitosis

doi: 10.7554/eLife.44700

Figure Lengend Snippet: ( a ) Top left, schematic indicating the approximate cell cycle stages separated by Cyclin B1 and phospho-Histone H3 (Ser10) antibody labeling. Bottom, representative FACS scatter plots indicating the separation of early (prophase to metaphase) and late (anaphase to telophase) mitotic L1210 cells. Arresting cells to metaphase using STLC increases the number of cells in early mitosis but decreases the number of cells in late mitosis, validating the cell cycle separation. Top right, a histogram indicating O-propargyl-puromycin (OPP) protein synthesis assay specificity, by comparing control (orange) and 100 µM cycloheximide (CHX) (blue) treated samples. (n = 6 separate cultures for control, three separate cultures for Blank, CHX and STLC samples). ( b ) Representative image of control L1210 cells labeled with OPP and phospho-Histone H3 (Ser10) antibody (n = 12 fields of view). An early mitotic cell (white arrow), with phospho-Histone H3 (Ser10) labeling but not fully compacted chromatin, displays OPP incorporation comparable to OPP incorporation in non-mitotic cells (cells without arrows). A metaphase cell (pink arrowhead), with phospho-Histone H3 (Ser10) labelling and compacted chromatin, displays low OPP incorporation. Dashed yellow lines indicate cell outlines. ( c ) Protein synthesis rates of G2, early mitotic and late mitotic L1210 cells. Early and late mitotic cells were separated as shown in ( a ). Chemical treatment with 5 µM STLC lasted 2 hr and with 100 µM CHX lasted 30 min. Data in late mitosis after STLC treatment is indicated with light green bar, as this population had too few cells for a reliable analysis. (n = 3–6 separate cultures). ( d ) Protein synthesis rates of G2 and mitotic L1210 cells after 4 hr treatment with 100 nM CHX. (n = 4 separate cultures). In ( c ) and ( d ), data depicts mean ± SD. p-Values obtained using ANOVA followed by Tukey’s posthoc test.

Article Snippet: Antibody , Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit monoclonal Ab (Alexa Fluor 488 Conjugate) , Cell Signaling Technology , Cat#3465; RRID: AB_10695860 , Dilution 1/100 in PBS supplemented with 5% BSA.

Techniques: Antibody Labeling, Control, Labeling

Journal: eLife

Article Title: Mammalian cell growth dynamics in mitosis

doi: 10.7554/eLife.44700

Figure Lengend Snippet:

Article Snippet: Antibody , Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit monoclonal Ab (Alexa Fluor 488 Conjugate) , Cell Signaling Technology , Cat#3465; RRID: AB_10695860 , Dilution 1/100 in PBS supplemented with 5% BSA.

Techniques: Generated, Transfection, Construct, shRNA, Isolation, Control, Activity Assay, Software

Journal: Development (Cambridge, England)

Article Title: Drosophila Cks30A interacts with Cdk1 to target Cyclin A for destruction in the female germline.

doi: 10.1242/dev.01940

Figure Lengend Snippet: Fig. 2. Female meiosis is disrupted in rem mutants. (A,B,D,F) remHG24/ remHG24, (C,E,G) wild type and (H) remRA74/Df(2L)30AC, labeled for DNA (green) and microtubules (red). (A) Metaphase I of meiosis is typically not affected in rem mutants, but 7% display disorganized spindles as in B. (C,D) Metaphase of meiosis II. In wild type, the meiosis II spindles are arrayed in tandem and connected by the spindle mid-body (arrow), while in rem, the spindles are kinked or separated. (E,F) Anaphase II. In wild type, four meiotic products with equal DNA content can be distinguished. rem spindles frequently break down and mis-segregation occurs. In the lower spindle, the two meiotic products are of different sizes, while the upper spindle has not entered anaphase. Pronuclear fusion in wild type (G) and rem (H). All nuclei are in interphase, and the male pronucleus and female pronucleus are in apposition and associated with the male sperm aster (arrows in G,H). The male pronucleus is largely obscured by the sperm aster in H. (I,J) Polar bodies from wild type (I) and remHG24/remRA74 (J) stained for microtubules (red) and the prophase/metaphase marker phospho-histone H3 (blue). Overlap appears pink. In wild type and in rem, DNA labels with phospho-histone H3, although in rem the chromatin is surrounded by microtubules within a shell, while in wild type the chromatin radiates out from a microtubule array. Scale bar: in A, 5 μm for A-H.

Article Snippet: Antibodies were rat α-Tubulin (Cappel) at 1/200, rabbit α-Cnn (gift of Thomas Kaufman) at 1/500, mouse α-Lamin Dmo (Development Studies Hybridoma Bank) at 1/20, rabbit αphospho-histone H3 (Upstate) at 1/500, rabbit α-Caspase 3 (Cell Signaling Technologies) at 1/500.

Techniques: Labeling, Staining, Marker