brdu dna content flow cytometry primary fibroblasts  (Thermo Fisher)

Journal: Genes & Development

Article Title: PRIM1 deficiency causes a distinctive primordial dwarfism syndrome

doi: 10.1101/gad.340190.120

Figure Lengend Snippet: The c.103+1G>T and C301R variants reduce PRIM1 protein levels. (A) Cysteine 301, substituted to arginine in P5, lies in a buried hydrophobic region. DNA primase dimer crystal structure (PDB: 4BPU) with residues shaded according to solvent accessibility. (B) Schematic of the FACS-based dual-reporter stability assay. Expression vector expresses an mRNA encoding PRIM1-GFP-P2A-FLAG-SR-P2A-RFP. Intervening P2A “self-cleaving” peptide sequences produce PRIM1-EGFP, FLAG-SR, and RFP polypeptides in equimolar amounts. PRIM1-GFP (wild type and mutants) and RFP levels are assayed in individual cells by flow cytometry. PRIM1-GFP and FLAG-SR levels can be independently assessed by immunoblotting (see Supplemental Fig. S3D). (C) GFP-RFP scatter plot for wild-type, C301R, and V35insDGV dual reporter constructs. n = WT, 27,027; C301R, 44,863; V35insDGV 46,441 cells respectively. (rfu) Relative fluorescence units. (D) Kernel density estimation plot of GFP:RFP ratios from C. (E) Schematic depicting the consequence of the c.103+1G>T variant on splicing. Reference and alternate sequences of intron 1 are shown with positions of the reference and cryptic splice donor sites marked by dotted lines. SpliceAI scores for splice donor sites in brackets. (Red box) Nine nucleotides of intron 6 included as a result of activation of the cryptic splice donor variant. (F) Schematic assaying the effect of the c.103+1G>T variant. Minigene assay. DNA spanning exon 1 (Ex1) and exon 2 (Ex2) of PRIM1 was cloned into the minigene. (Arrows) Position of PCR primers, (dotted line) splicing from cryptic splice donor. (G) Representative cDNA Sanger sequence traces from wild-type (WT) and c.103+1G>T variant minigene constructs. The three-amino-acid (DGV) insertion from the c.103+1G>T variant marked in red.

Article Snippet: BrdU-DNA content flow cytometry Primary fibroblasts were seeded into AmnioMAX medium (Life Technologies) to achieve ∼60% confluency after 24 h. Cells were then incubation with 10 μM BrdU for 30 min, washed, harvested, and fixed with 70% EtOH for 16 h at −20°C.

Techniques: Solvent, Stability Assay, Expressing, Plasmid Preparation, Flow Cytometry, Western Blot, Construct, Fluorescence, Variant Assay, Activation Assay, Mini Gene Assay, Clone Assay, Sequencing

Journal: Genes & Development

Article Title: PRIM1 deficiency causes a distinctive primordial dwarfism syndrome

doi: 10.1101/gad.340190.120

Figure Lengend Snippet: Reduced cell proliferation and impaired DNA replication in PRIM1-deficient primary fibroblasts. (A) Cell doubling time plotted for three independent experiments on P3 and two unrelated control C1 and C2 primary fibroblast cell lines. Bars indicate the mean. Error bars indicate SD. (B) Schematic of CldU/IdU double-pulse experiment used to determine S-phase time. Cells were labeled with CldU at t = 0, followed by IdU after 1.5 h. Cells leaving S phase (Lcells) are labeled with CldU only, while cells remaining in S phase (Scells) are labeled with both CldU and IdU. (Ts) S-phase length, the product of interval between pulses (Ti) and the proportion of Scells to Lcells (Martynoga et al. 2005). (C) S-phase time is substantially increased in P3 fibroblasts compared with controls. Mean ± SEM, N = 3 experiments. (D) Schematic of rescue experiment. P3 fibroblast transfected with either empty vector (EV), wild-type (WT) or C301R-PRIM1-GFP as indicated and after 24 h S-phase time determined as in B for GFP + ve cells. (E) Complementation with WT PRIM1-GFP rescues slow S-phase progression in P3 fibroblasts. S-phase length plotted for n = 3 experiments. Mean ± SEM. (F) DNA content and BrdU flow cytometry scatter plots, representative of four independent experiments on control (C1 and C2) and P3 primary fibroblast cell lines. (G) BrdU incorporation is reduced in PRIM1-deficient cells during S-phase. Quantification of BrdU mean fluorescence intensity (MFI) from control and patient-derived fibroblasts according to S-phase gate in F. (a.u.) Arbitrary units. (H) γ-H2AX is increased in S-phase P3 fibroblasts. Mean γ-H2AX intensity calculated for EdU-positive nuclei from C1, C2, and P3 cells. n = 3 experiments. Data points are colored by experiment. (Filled circles) Mean values for each replicate, (bars) median and interquartile range (all values). Values were normalized for each experiment relative to C1 mean value. (P-values) Repeat measures ANOVA with Tukey multiple comparison test. (I) Representative immunofluorescence images of S-phase nuclei quantified in H. Scale bar, 5 µm. Statistics in A, C, E, and G are one-way ANOVA with Tukey multiple comparison test.

Article Snippet: BrdU-DNA content flow cytometry Primary fibroblasts were seeded into AmnioMAX medium (Life Technologies) to achieve ∼60% confluency after 24 h. Cells were then incubation with 10 μM BrdU for 30 min, washed, harvested, and fixed with 70% EtOH for 16 h at −20°C.

Techniques: Control, Labeling, Transfection, Plasmid Preparation, Flow Cytometry, BrdU Incorporation Assay, Fluorescence, Derivative Assay, Comparison, Immunofluorescence