Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Microglia in the developing retina couple phagocytosis with the progression of apoptosis via P2RY12 signaling

doi: 10.1002/dvdy.163

Figure Lengend Snippet: A-D. Each circle is color coded for each embryo imaged, and overlaid onto box plots. A. Total number of AO+ cells observed during live imaging from ~54-62 hours post fertilization (hpf). B. The percent of AO+ cells that were observed to be engulfed by a microglial cell during the imaging session. C. For AO+ cells observed to be cleared by microglia, the percent showing association with a microglial cell at the onset of AO+ signal. D. Each circle represents individual cells from each embryo that was imaged. A selected number of individual AO+ cells, which were observed to appear and be cleared, during the imaging session were tracked in each embryo imaged. The duration of AO signal is shown for these individual AO+ cells. E-F represent max projections of selected, flattened z planes over a selected time-lapse. E. Shows the initial detection of an AO+ cell (green, indicated by red-orange arrow) associated with a microglial cell (microglia=magenta). E’. Shows the same AO+ cell (green, indicated by the magenta arrow) 80 minutes later, just before the AO signal is lost, after it had moved with the migrating microglial cell (magenta). F. The movement of the AO+ cell body was temporally color-coded to show its dynamic movement over time; the AO+ cell was associated with a microglial cell at all time frames. The color scale on the right indicates progression of time, where the first frame corresponds to 0:00 and the last frame is 85:00 (min:sec). Scale bar in bottom right of F = 5 microns and applies to E-F. G and H. To show a representation of dynamics of AO+ cell movement in association with microglia, a subset of AO+ cells were tracked in 3D using Imaris software. Quantifications show the speed (G) and total displacement (H) of individual AO+ cell bodies. Box plots are shown and circles are color coded to represent each embryo imaged. Each circle represents one AO+ cell that was tracked in each embryo that was imaged. Results are shown for n=6 different embryos.

Article Snippet: Scale bar in bottom right of F = 5 microns and applies to E-F. G and H. To show a representation of dynamics of AO+ cell movement in association with microglia, a subset of AO+ cells were tracked in 3D using Imaris software.

Techniques: Imaging, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Intratumoral oxygen gradients mediate sarcoma cell invasion

doi: 10.1073/pnas.1605317113

Figure Lengend Snippet: Minoxidil inhibits sarcoma cell migration and matrix remodeling in hypoxic hydrogel. KIA-GFP cells were tracked on day 3 in hypoxic-treated and untreated hydrogel to determine 3D trajectories of tracked cells (representative trajectories) (A); overall speed (B); velocity in the x, y, and z directions (C); and MSD in the x, y, and z directions (D). Plots were created using the position of KIA-GFP cells in the hydrogels. (E) Collagen deposition and quantification (collagen in red, nuclei in blue). (Scale bars: 50 μm.) (F) Proteolytic degradation of HI hydrogels incorporating DQ Gtn for 3 d. (Left) Representative fluorescence microscopy images. (Scale bars: 20 μm). (Right) Quantitative analysis of relative fluorescence intensity. (G) Western blot analyses for PLOD2 in KIA cells cultured in hypoxic conditions with and without minoxidil treatment. Significance levels: *P < 0.05; ^P < 0.01; #P < 0.001.

Article Snippet: Cells were tracked at day 3 using live-cell 3D confocal microscopy (LSM 780; Carl Zeiss) equipped with a cell incubator (5% CO 2 and 37 °C).

Techniques: Migration, Fluorescence, Microscopy, Western Blot, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Intratumoral oxygen gradients mediate sarcoma cell invasion

doi: 10.1073/pnas.1605317113

Figure Lengend Snippet: Minoxidil inhibits sarcoma cell migration and matrix remodeling in hypoxic hydrogel. KIA-GFP cells were tracked on day 3 in hypoxic-treated and untreated hydrogel to determine 3D trajectories of tracked cells (representative trajectories) (A); overall speed (B); velocity in the x, y, and z directions (C); and MSD in the x, y, and z directions (D). Plots were created using the position of KIA-GFP cells in the hydrogels. (E) Collagen deposition and quantification (collagen in red, nuclei in blue). (Scale bars: 50 μm.) (F) Proteolytic degradation of HI hydrogels incorporating DQ Gtn for 3 d. (Left) Representative fluorescence microscopy images. (Scale bars: 20 μm). (Right) Quantitative analysis of relative fluorescence intensity. (G) Western blot analyses for PLOD2 in KIA cells cultured in hypoxic conditions with and without minoxidil treatment. Significance levels: *P < 0.05; ^P < 0.01; #P < 0.001.

Article Snippet: Cells were tracked at day 3 using live-cell 3D confocal microscopy (LSM 780; Carl Zeiss) equipped with a cell incubator (5% CO 2 and 37 °C).

Techniques: Migration, Fluorescence, Microscopy, Western Blot, Cell Culture

Journal: ACS Bio & Med Chem Au

Article Title: Multiplex Protein Imaging through PACIFIC: Photoactive Immunofluorescence with Iterative Cleavage

doi: 10.1021/acsbiomedchemau.3c00018

Figure Lengend Snippet: Cell motility study combining live-cell tracking with PACIFIC. (a) Workflow of the experiment. (b) Cell movement trajectories of living cells without perturbation, with EGFR inhibition (erlotinib), and with AKT inhibition (AZD8055). (c) Scatter plot of protein expression levels and the confinement ratio extracted from the single cells. (d) t-SNE plot of the single-cell data set followed by nearest neighbor clustering to partition the single-cell data into phenotypical subpopulations. (e) Cell movement trajectories of living cells without perturbation, with p70S6K inhibition (LY2584702), and combinations of LY2584702 with EGFR or AKT inhibitors. (f) Scatter plot of confinement ratio extracted from live-cell tracking. (**, p < 0.01; ***, p < 0.001).

Article Snippet: Erlotinib, AZD8055, and LY2584702 were purchased from SelleckChem (Houston, TX).

Techniques: Cell Tracking Assay, Inhibition, Expressing

Journal: bioRxiv

Article Title: A parallel cell-cycle entry pathway with inverted G1 signaling and alternate point of no return

doi: 10.1101/600007

Figure Lengend Snippet: a ,Two paths to start the cell cycle. b , Left: Degron-based APC/C activity reporter. Right: Mitogen-starved MCF-10A cells expressing the APC/C activity reporter were imaged at the time of mitogen and 1μM Palbociclib (CDK4/6 inhibitor) addition. Time points taken every 12 minutes. CDK4/6 inhibitor refreshed at 24hrs. 1 of n=5 biological replicates. c , MCF-10A cells mitogen-released in the presence of 100nM EdU with or without CDK4/6 inhibitor. Cells were fixed after 24hrs (26838, 36549, 29371 cells for unreleased, DMSO, and CDK4/6 inhibitor; 1 of n=3 biological replicates). d , Cells were mitogen-released with or without indicated CDK4/6 inhibitor concentrations and the drug was refreshed every 12hrs, 24hrs, or not at all. Cells fixed at 36hrs after mitogen release. S/G2 cells determined via EdU incorporation and DNA content. Error bars: SEM from 3 biological replicates; p-values calculated using two-sided, two-sample t-tests. e , CDF of mitogen-released cells inactivating APC/C CDH1 . Only cells present at the time of mitogen-release are tracked. f , APC/C activity in cells born into DMSO or CDK4/6 inhibitor. Cells aligned at birth. 1 of n=3 biological replicates. g , Different cell lines treated with 1μM CDK4/6 inhibitor for 48hrs and refreshed at 24hrs. Percent S/G2 defined as >2n DNA (determined by Hoechst) and high endogenous geminin (an APC/C CDH1 substrate). Percentages normalized to the DMSO-treated condition. h , Generation comparison of cell-cycle re-entry percentages in MCF-10A (Rb intact) and HeLa (Rb inactivated) cells (error bars: SEM from 3 biological replicates; p-values calculated using two-sided, two-sample t-tests).

Article Snippet: The inhibitors used in this study were: CDK1 inhibitor RO3306 at 10μM (Sigma-Aldrich SML0569), CDK4/6 inhibitor PD0332991 (Palbociclib) at 1μM unless indicated otherwise (Selleck Chemicals S1116), CDK4/6 inhibitor LY2835219 (Abemaciclib) at 3μM (Selleck Chemicals S7158), CDK4/6 inhibitor LEE011 (Ribociclib) at 9μM (Selleck Chemicals S7440), and CDK1/2 inhibitor CAS 443798-55-8 at 3μM (EMD Biosciences #217714).

Techniques: Activity Assay, Expressing

Journal: bioRxiv

Article Title: A parallel cell-cycle entry pathway with inverted G1 signaling and alternate point of no return

doi: 10.1101/600007

Figure Lengend Snippet: a , DHB-based cyclin E/A-CDK activity reporter. b , Single-cell traces of cyclin E/A-CDK activity after mitogen release with or without 1μM CDK4/6 inhibitor (100 cells plotted; 2782 and 8538 cells total for DMSO and CDK4/6 inhibitor; 1 of n=2 biological replicates). Activity baseline at time 0 is determined to be at most 0.7. Blue: >0.7 at last time point. Green: <0.7 at last time point, exceeded 0.7 at some point between 8 and 24hrs. Bottom: Examples of fluctuating cyclin E/A-CDK activity. c , Top: immunofluorescence validation of cyclin E1 and p21 knockdown. Bottom: effect of knockdown on cyclin E/A-CDK activity in cells treated with CDK4/6 inhibitor. Traces classified the same way as b (100 cells plotted; 2958, 3707, 3113 cells for si Ctrl, si Cyclin E1, and si p21; 1 of n=2 biological replicates). d , Cells were treated with neocarzinostatin (NCS) for 15min, and then mitogen-released for 24hrs. Percent cells with high cyclin E/A-CDK activity (defined as reporter ratio>0.7) are displayed. Percentages normalized to 0ng/mL condition. Error bars: standard deviation of 2 biological replicates. e , Cells were stably infected with DHFR-Myc that is unstable until TMP molecule addition. c-Myc levels were induced to ~50% above control after adding 10μM TMP at the time of mitogen release. Cells fixed at 24 and 48hrs after mitogen release. Drugs refreshed every 24hrs. Error bars: SEM from 3 biological replicates; p-values calculated using two-sided, two-sample t-tests. f , Left: immunofluorescence of c-Myc knockdown validation (error bars: SEM from 3 biological replicates). Right: cyclin E/A-CDK activity after mitogen-release in MCF-10A (100 cells plotted, 569, 3446, 3904, 3935, 3767 total cells for unreleased, si Ctrl+DMSO, si Ctrl+CDK4/6 inhibitor, si c-Myc+DMSO, and si c-Myc+CDK4/6 inhibitor, respectively), representative of n=5 biological replicates.

Article Snippet: The inhibitors used in this study were: CDK1 inhibitor RO3306 at 10μM (Sigma-Aldrich SML0569), CDK4/6 inhibitor PD0332991 (Palbociclib) at 1μM unless indicated otherwise (Selleck Chemicals S1116), CDK4/6 inhibitor LY2835219 (Abemaciclib) at 3μM (Selleck Chemicals S7158), CDK4/6 inhibitor LEE011 (Ribociclib) at 9μM (Selleck Chemicals S7440), and CDK1/2 inhibitor CAS 443798-55-8 at 3μM (EMD Biosciences #217714).

Techniques: Activity Assay, Immunofluorescence, Standard Deviation, Stable Transfection, Infection

Journal: bioRxiv

Article Title: A parallel cell-cycle entry pathway with inverted G1 signaling and alternate point of no return

doi: 10.1101/600007

Figure Lengend Snippet: a , E2F is active when Rb is hyperphosphorylated. b , Phospho-Rb analysis after live-cell tracking of cyclin E/A-CDK activity. Scale bar=10μm. c , Phospho-Rb(S807/S811) analysis of cells that have inactivated APC/C CDH1 (top; 50 cells plotted, 2315 cells total), and cells that have initiated cyclin E/A-CDK activity and have not inactivated APC/C CDH1 (bottom; 50 cells plotted, 364, 1023 cells for DMSO and CDK4/6 inhibitor). 1 of n=3 biological replicates. d , BJ-5ta cells expressing the cyclin E/A-CDK activity reporter and APC/C degron reporter were mitogen-released with or without 1μM CDK4/6 inhibitor, fixed after 24hrs, and stained for Rb and phospho-Rb(S807/S811). G1 cells with recently activated cyclin E/A-CDK activity were analyzed. e , Cells were grouped into high and low phospho-Rb(S807/S811) signal populations. The percentages of cells with high signal were then determined for each bin of the cyclin E/A-CDK activity. Middle: Cells born into DMSO or CDK4/6 inhibitor. Error bars denote standard deviation of 3 biological replicates. Right: Cells mitogen-released with DMSO or CDK4/6 inhibitor. Error bars denote standard deviation of 2 biological replicates. f , Asynchronously cycling cells treated with or without CDK4/6 inhibitor for 24hrs. Cells were then incubated with 10μM EdU for 15min, and then treated with DMSO, CDK1/2i (3μM), or CDK1i (10μM) for 20min. Cells were then fixed and high EdU signal cells were examined. Data points denote biological replicates.

Article Snippet: The inhibitors used in this study were: CDK1 inhibitor RO3306 at 10μM (Sigma-Aldrich SML0569), CDK4/6 inhibitor PD0332991 (Palbociclib) at 1μM unless indicated otherwise (Selleck Chemicals S1116), CDK4/6 inhibitor LY2835219 (Abemaciclib) at 3μM (Selleck Chemicals S7158), CDK4/6 inhibitor LEE011 (Ribociclib) at 9μM (Selleck Chemicals S7440), and CDK1/2 inhibitor CAS 443798-55-8 at 3μM (EMD Biosciences #217714).

Techniques: Cell Tracking Assay, Activity Assay, Expressing, Staining, Standard Deviation, Incubation

Journal: bioRxiv

Article Title: A parallel cell-cycle entry pathway with inverted G1 signaling and alternate point of no return

doi: 10.1101/600007

Figure Lengend Snippet: a , Cells mitogen-released with DMSO or 1μM CDK4/6 inhibitor and have recently inactivated APC/C CDH1 were analyzed for phospho-Rb(S807/S811) signal (>800 cells per histogram for DMSO, >200 cells per histogram for CDK4/6 inhibitor; 1 of n=3 biological replicates). b , Top: Single-cell mRNA quantification (scale bar=10μm). Bottom: E2F1 mRNA in cells released with or without CDK4/6 inhibitor. High cyclin E/A-CDK activity defined as ratio of signal>0.7. p-values calculated using two-sided, two-sample t-tests (from left to right: n=784, 1484, 638, and 98 cells). 1 of n=3 biological replicates. c , Mitogen-released cells were assayed for chromatin-bound Rb. Green arrows: cells that have inactivated both Rb and APC/C CDH1 ; blue arrows: cells that have inactivated Rb but not APC/C CDH1 ; orange arrows: cells that have inactivated APC/C CDH1 but not Rb. Scale bar: 10μm. 1 of n=2 biological replicates. d , Experiment setup for drugging mice. e , Illustration of proliferative and post-mitotic cells in the small intestinal crypt. f , Mice small intestinal crypts that were treated with vehicle or CDK4/6 inhibitor. The cells express the APC/C activity reporter and were stained for phospho-Rb (S807/S811). Blue arrows denote example cells with high phospho-Rb and low APC/C degron signal. Orange arrows denote cells with high APC/C degron and low phospho-Rb signal. Crypts are oriented in an U-shape.

Article Snippet: The inhibitors used in this study were: CDK1 inhibitor RO3306 at 10μM (Sigma-Aldrich SML0569), CDK4/6 inhibitor PD0332991 (Palbociclib) at 1μM unless indicated otherwise (Selleck Chemicals S1116), CDK4/6 inhibitor LY2835219 (Abemaciclib) at 3μM (Selleck Chemicals S7158), CDK4/6 inhibitor LEE011 (Ribociclib) at 9μM (Selleck Chemicals S7440), and CDK1/2 inhibitor CAS 443798-55-8 at 3μM (EMD Biosciences #217714).

Techniques: Activity Assay, Staining

Journal: bioRxiv

Article Title: A parallel cell-cycle entry pathway with inverted G1 signaling and alternate point of no return

doi: 10.1101/600007

Figure Lengend Snippet: a , Cells born into DMSO or 1μM CDK4/6 inhibitor were treated with 10μM EdU for 15min, and then assayed for EdU and phospho-Rb(S807/S811) (5000 cells plotted, 1 of n=3 biological replicates). b , Mitogen-released cells treated with DMSO or CDK4/6 inhibitor were measured for percent cells with high phospho-Rb(S807/S811) signal, high APC/C degron, and high EdU signal (after 15min of 10μM EdU treatment). Error bars: SEM from 3 biological replicates. c , EMI1 ensures irreversible APC/C CDH1 inactivation. d-h , Cells born into DMSO or CDK4/6 inhibitor and have inactivated APC/C CDH1 (with the exception of f , where it is just cells born into DMSO or CDK4/6 inhibitor) were treated with 3μM CDK1/2i ( d-e ) or an extra 100mM salt ( f-h ). Dashed lines denote 25 th and 75 th percentile. Gray boxes denote conditions where cells re-activated APC/C CDH1 . n=2 biological replicates; >30 cells per condition. In g-h , to enrich for Rb inactivated cells in the 6hrs post APC/C CDH1 inactivation conditions, only cells with cyclin E/A-CDK>0.8 right before treatment were analyzed (threshold determined from ).

Article Snippet: The inhibitors used in this study were: CDK1 inhibitor RO3306 at 10μM (Sigma-Aldrich SML0569), CDK4/6 inhibitor PD0332991 (Palbociclib) at 1μM unless indicated otherwise (Selleck Chemicals S1116), CDK4/6 inhibitor LY2835219 (Abemaciclib) at 3μM (Selleck Chemicals S7158), CDK4/6 inhibitor LEE011 (Ribociclib) at 9μM (Selleck Chemicals S7440), and CDK1/2 inhibitor CAS 443798-55-8 at 3μM (EMD Biosciences #217714).

Techniques: