Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A . Schematic of inducible H2B-FT expression construct. B. Representative images of HeLa cells expressing the H2B-FT. C. Selected time series of H2B-FT fluorescence in HeLa cells from a representative field of view following doxycycline (Dox) induction. D-E . Quantification of average nuclear blue (D) and red (E) FT intensity over time following Dox treatment. F. Selected time series of HeLa cells after Dox washout at T=4.5 h. G . Data from (D-E) plotted as blue/red ratio. Error bars represent standard deviation across n = 3 culture wells. Nuclear intensity measurements were normalized to timepoint 0. Scale bars = 50µm.

Article Snippet: The H2B-FT fusion transgenes were constructed by overlap extension PCR using the three FT plasmids as well as the human H2B.J coding sequence from PGK-H2B-mCherry (Addgene # 21217) as templates, and cloned into the pFU-TetO-Gateway-PGK-Puro and pSCMV expression plasmids.

Techniques: Expressing, Construct, Fluorescence, Standard Deviation

Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A. Changes in cell number over time in primary iH2B-FT MEFs following transduction with either c-Myc or empty vector control (EV). Error bars denote standard deviation across n=4 culture wells. B. The ratio of blue/red fluorescence intensity was determined for individual cells at 25h and 70h post transduction. Box plots represent the median and interquartile range and whiskers represent 5th-95th percentile. P=0.295 (25h) and P<0.0001 (70h), determined using Mann-Whitney test with a 99% confidence interval. C. The blue and red fluorescence level of individual cells at 70h post transduction. Each dot denotes a single cell. FACS-style gates were applied to representative scatter plots. D. The percentage of cells within each gate. Error bars show standard deviation across n=4 culture wells. P=0.0035 (Red Cells) and P=0.0498 (Blue Cells), determined using Student’s T-Test with Welch’s correction, 95% confidence interval. dF=4.057 (Red Cells) and dF=3.221 (Blue Cells). E. The cell cycle lengths of individual cells from each gate were determined by image tracking. Cell cycle length represents the time interval between two consecutive mitoses within the same cell lineage. Cell cycle length is heterogeneous and the distribution of cell cycle lengths for each condition are shown as histograms. n values refer to the number of cells tracked for each condition. F. The relationship between cell cycle length and H2B-FT blue/red ratio. Each dot denotes an individual cell. All trackable cells (n=189) from both conditions are plotted. Spearman correlation coefficient = −0.7808. P<0.0001 was calculated with a 95% confidence interval.

Article Snippet: The H2B-FT fusion transgenes were constructed by overlap extension PCR using the three FT plasmids as well as the human H2B.J coding sequence from PGK-H2B-mCherry (Addgene # 21217) as templates, and cloned into the pFU-TetO-Gateway-PGK-Puro and pSCMV expression plasmids.

Techniques: Transduction, Plasmid Preparation, Standard Deviation, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A. BaF3 cells proliferate at different rates in varying IL-3 concentrations, as determined by cell counting. Error bars denote standard deviation across n=6 culture wells. B. Representative FACS plots of BaF3 cells expressing the H2B-FT reporter grown under different IL-3 concentrations. C. Histograms of blue/red fluorescence ratio derived from FACS data in (B). D. Representative colony morphology and FT fluorescence of H2B-FT-Medium knock-in mESCs maintained in feeder-free conditions. E. Confirmation of cell cycle change following 48h of RA treatment, as analyzed by EdU pulse-labeling and DNA content profiling. F. Representative FACS plots of red vs. blue fluorescence in pluripotent and RA-treated mESCs (top). These FACS data were re-plotted as histograms of blue/red ratio (bottom). G-H. Pluripotent and RA-treated mESCs stably transfected with H2B-FT via Sleeping Beauty transposon were FACS sorted according to their blue/red FT ratio. Representative gating strategy is shown. H2B-FT-negative gate was determined using non-transfected WT mESCs. I. DAPI/EdU cell cycle profiles of sub-populations sorted from mESCs and RA-treated cells. J. Frequency of cells in G1 vs. G2/M from the FACS-sorted populations shown in (I). K. The number of alkaline phosphatase positive colonies formed by the same number of cells sorted in (G) following 6 days of culture. Error bars denote standard deviation across n=3-4 culture wells. Significance determined using Student’s T-Test with a 95% confidence interval, df=6 (mESCs) and df=5-6 (RA 48h). Exact P values provided in Table S3. Scale bar in (D) = 80µm.

Article Snippet: The H2B-FT fusion transgenes were constructed by overlap extension PCR using the three FT plasmids as well as the human H2B.J coding sequence from PGK-H2B-mCherry (Addgene # 21217) as templates, and cloned into the pFU-TetO-Gateway-PGK-Puro and pSCMV expression plasmids.

Techniques: Cell Counting, Standard Deviation, Expressing, Fluorescence, Derivative Assay, Knock-In, Labeling, Stable Transfection, Transfection

Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A. Representative FACS plots of blue/red profile in LKS and GMP cells from reconstituted mice 2 months after transplantation with HSPCs virally expressing the H2B-FT reporter. B. Distribution of blue/red ratio in H2B-FT-expressing LKS and GMP populations, overlaid on that of whole bone marrow. C. FACS plots of myeloid (Mac1+) and erythroid (Ter119+) cells from reconstituted mouse bone marrow. D. Data from (C) plotted as histograms of blue/red H2B-FT ratio. Gates in (A) and (C) were used to exclude the non-transduced (H2B-FT-negative) cells. E. Targeting strategy for the HPRT:: TetO-H2B-FT mouse allele. F. DAPI/EdU cell cycle profiles of GMPs vs. early erythroid cells following 2 hours of in vivo EdU labeling. G. Distributions of H2B-FT blue/red ratio in LKS, GMPs, and early erythroid cells. H. Frequency of myeloid (Mac1+), B-cell (B220+), and T-cell (CD3+) lineages in peripheral blood of healthy H2B-FT knock-in mice vs. those crossed with MLL-ENL (n=3 mice per group). All mice were treated with Dox for at least 8 days prior to analysis. Error bars represent standard deviation. P=0.0090 (Mac1+), P=0.0072 (B220+), and P=0.7629 (CD3+) determined using Student’s T-Test with a 95% confidence interval, dF=4. I. Representative H2B-FT blue/red profiles within defined peripheral blood subsets as in (H).

Article Snippet: The H2B-FT fusion transgenes were constructed by overlap extension PCR using the three FT plasmids as well as the human H2B.J coding sequence from PGK-H2B-mCherry (Addgene # 21217) as templates, and cloned into the pFU-TetO-Gateway-PGK-Puro and pSCMV expression plasmids.

Techniques: Transplantation Assay, Expressing, In Vivo, Labeling, Knock-In, Standard Deviation

Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A. 5µm frozen sections from kidney and pylorus in two orientations of an adult iH2B-FT reporter mouse were scanned by microscopy to capture blue and red fluorescence. Gastric glands are shown in elongated orientation (pylorus I) and oblique/cross-sectional orientation (pylorus II). Bottom, the neocortex of a representative E17.5 iH2B-FT mouse embryo. L= pyloric lumen; S=submucosa. Dotted line in bottom left image follows the line of a cerebral lateral ventricle. B. The blue/red ratio for each image shown in (A) was quantified and plotted as a histogram, from which five populations of increasing blue/red ratio (P1-P5) were determined. C. Left, a heatmap corresponding to populations P1-P5 as shown in (B) was applied onto the original images (A) to indicate the relative locations occupied by cells of different blue/red ratios. Right, the same regions as imaged after fixation and DAPI/EdU labeling. EdU was injected 35 minutes before harvesting mice/embryos. D. Magnified detail of white boxed pyloric regions shown in (C). White arrow indicates the base of a gastric gland. E. Tiled images showing a zoomed-out view of an E17.5 embryonic brain hemisphere. Left, H2B-FT blue/red merged image. Middle, heatmap of blue/red ratio binned from histograms as in (B). Right, same region imaged after DAPI/EdU labeling. Dotted line indicates cerebral lateral ventricle; IZ=intermediate zone, CP=cortical plate, MZ=marginal zone, Th=thalamus. F. Blue/red ratio histograms from all three tissues overlaid on the same plot. “Pylorus” histogram includes combined data from both images of the pylorus (middle images, A). All images contained within a single panel are shown at the same scale. Scale bar in (A,C) = 200µm. Scale bar in (D) = 80µm. Scale bar in (E) = 500µm.

Article Snippet: The H2B-FT fusion transgenes were constructed by overlap extension PCR using the three FT plasmids as well as the human H2B.J coding sequence from PGK-H2B-mCherry (Addgene # 21217) as templates, and cloned into the pFU-TetO-Gateway-PGK-Puro and pSCMV expression plasmids.

Techniques: Microscopy, Fluorescence, Labeling, Injection

Journal: bioRxiv

Article Title: Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

doi: 10.1101/494252

Figure Lengend Snippet: A. Cell cycle length distribution of cultured GMPs. Individual cell cycle length was determined by time-lapse microscopy. Dotted lines show the indicated percentiles. B. H2B-FT blue/red ratio distribution of the same GMPs as analyzed by flow cytometry after 24 hours culture. C. Calculated cell cycle length distributions of designated bone marrow populations from a representative H2B-FT mouse. D. Cell cycle length distribution of normal GMPs and L-GMPs from a representative mouse induced to express MLL-ENL. E. Median cell cycle length variation among three H2B-FT mice, shown by percent coefficient of variation (CV%). F. Cell cycle length distributions of LKS and GMP populations from the three mice. Boxes in panels (E), (F), and (H) represent the median and interquartile range of each group; whiskers represent 5th-95th percentile.

Article Snippet: The H2B-FT fusion transgenes were constructed by overlap extension PCR using the three FT plasmids as well as the human H2B.J coding sequence from PGK-H2B-mCherry (Addgene # 21217) as templates, and cloned into the pFU-TetO-Gateway-PGK-Puro and pSCMV expression plasmids.

Techniques: Cell Culture, Time-lapse Microscopy, Flow Cytometry

Journal: bioRxiv

Article Title: An shRNA screen in primary human beta cells identifies the serotonin 1F receptor as a negative regulator of survival during transplant

doi: 10.1101/2024.05.01.591950

Figure Lengend Snippet: (A) Static glucose stimulated insulin secretion in presence of vehicle or 150nM of 1-(2-hydroxy-3-(naphthalen-2-yloxy)propyl)-4-(quinolin-3-yl)piperidin-4-ol. n=9 donors, 5 replicates for each donor. 2-way ANOVA: drug effect p<0.0001, glucose p<0.0001, interaction p=0.038. Post-hoc test p-values are Benjamini-Hochberg corrected. (B) Glucagon levels measured from a subset of (A), n=3 donors, 5 replicates for each donor. 2-way ANOVA: drug effect p<0.001, glucose p=0.2925, interaction p=0.3461. Post-hoc test p-values are Benjamini-Hochberg corrected. (C) Schematic of an insulin nanoluciferase BFP adenovirus to monitor insulin secretion from human beta cells. B = B chain of insulin, A = A chain of insulin, C = C-peptide. T2A = ribosomal skip sequence. BFP = blue fluorescent protein. (D) Correlation between insulin determined by ELISA and nanoluciferase luminescence from secretion samples of human islets infected with the insulin nanoluciferase reporter adenovirus shown in part C. n=9 independent samples from one human donor. (E) Image of human islets infected with adenovirus expressing control or 5-HT 1F shRNA mCherry co-infected with the insulin nanoluciferase BFP adenovirus and percent infection of co-infection, n=2 donors, 3-6 replicates for each donor (F) Human islets infected with the insulin nanoluciferase BFP adenovirus (MOI 50) and the 5-HT 1F shRNA or control shRNA adenovirus (MOI 200). 72 hours later, GSIS was performed. n=2 donors with 5 replicates for each donor. 3 way ANOVA 1-(2-hydroxy-3-(naphthalen-2-yloxy)propyl)-4-(quinolin-3-yl)piperidin-4-ol effect p=0.1077, glucose p<0.0001, and 5-HT 1F shRNA p=0.0392. Post-hoc test p-values were Benjamini-Hochberg FDR corrected. Error bars show standard error.

Article Snippet: GloSensor, cAMP-22p was cloned into pEN-RIP1-BGH-pA vector with T2a-h2b-mCherry between the EcoRI and XhoI by HiFi assembly (NEB, E2621).

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Control, shRNA