Journal: Oncogene

Article Title: Hypoxia-dependent recruitment of error-prone DNA polymerases to genome replication

doi: 10.1038/s41388-024-03192-0

Figure Lengend Snippet: A MCF7 cells were transfected with the indicated siRNA (50 nM) and incubated for 48 h. After that, the cells were exposed to hypoxia (0.5% oxygen) for 16 h, treated with BrdU for two hours, and fixated with ethanol. BrdU incorporation was analyzed using Roche’s ELISA, BrdU (colorimetric) kit according to manufacturer’s protocol. B MEFs with either a Pcna-K164R mutation or WT PCNA, were grown under hypoxia, and BrdU incorporation was measured as described in A . C XP12RO human cells were treated with the indicated siRNAs, followed by hypoxia and then viability analysis using the CellTiter-Glo® Luminescence Assay. D , E HEK293FT cells were subjected to 16 h hypoxia (0.5% oxygen) D or transfected with HA-HIF1A P402A/P564A under normoxia E , and the iPOND protocol was then conducted. In short, the cells were supplemented with EdU or DMSO, and after 60 min were crosslinked, permeabilized, and biotin was added for a click reaction with the EdU. The cells were lysed, and Streptavidin beads were added to capture the biotin. After a wash, the beads were boiled in an elution buffer, and the samples were analyzed by mass spectrometry. Hypoxia-specific enrichments of proteins on nascent DNA D was calculated from the MS results (Data Set 1) as: (Hypoxia Pulse/Chase LFQ-intensity ratio)/(Normoxia Pulse/Chase LFQ-intensity ratio). HIF1α-specific enrichments of proteins on nascent DNA E was calculated from the MS results (Data Set 2) as: (HIF1α Pulse/Chase LFQ-intensity ratio)/(Empty Vector Pulse/Chase LFQ-intensity ratio). The two datasets were deposited in the Figshare Repository at 10.6084/m9.figshare.27061339. The lists were filtered for proteins identified based on ≥10 unique peptides with ≥10% unique sequence coverage, and ranked in descending protein enrichment ratio. The 10 highest ranking proteins enriched on nascent DNA under hypoxia D or in normoxic cells expressing stable HIF1α E are presented.

Article Snippet: The Cell Proliferation ELISA, BrdU (colorimetric) kit (Merck) was used for measuring BrdU incorporation.

Techniques: Transfection, Incubation, BrdU Incorporation Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis, Luminescence Assay, Mass Spectrometry, Pulse Chase, Plasmid Preparation, Sequencing, Protein Enrichment, Expressing

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: An Orally Efficacious Thyrotropin Receptor Ligand Inhibits Growth and Metastatic Activity of Thyroid Cancers

doi: 10.1210/clinem/dgae114

Figure Lengend Snippet: Dose-dependent inhibition of cell proliferation by A35. The cells were treated with A35 for 72 or 96 hours. The filled circles (solid line) and filled squares (dashed line) represent the dose–response curve for A35 in MDA-T32 and MDA-T85 cells, respectively. Control (without A35) was set at 100%. Data are presented as mean ± SEM of at least 2 experiments with n = 4. Nonlinear 4 parameters variable slope fitting curves with 95% CI were drawn to determine the log(inhibitor, A35) vs response. (A) The number of viable cells, presented as the percentage of control, was dose-dependently decreased in both MDA-T32 and MDA-T85 treated with A35 and counted in Vi-CELL XR Cell Viability Analyzer at 72 hours post-treatment. (B) Cells were treated with A35 for 72 hours and allowed to incorporate BrdU for the final 4 hours. The OD values were plotted as the percentage of control. (C) Cell lines were treated with A35 for 72 to 96 hours, and WST-1 reagent was added to estimate cell proliferation/viability for the final 3 hours. The activity of A35 was measured spectrophotometrically for the formazan formed as a result of cell viability. The OD values are plotted as the percentage of control.

Article Snippet: To complement the studies of the effect of A35 on cell proliferation, BrdU (bromodeoxyuridine) incorporation into DNA was measured in the MDA-T32 and MDA-T85 cell lines using the Cell Proliferation ELISA, BrdU (colorimetric) kit from Millipore-Sigma (Roche, Cat# 11 647 229 001, RRID: AB_2922996).

Techniques: Inhibition, Control, Activity Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Cellular turnover and degradation of the most common missense cystathionine beta‐synthase variants causing homocystinuria

doi: 10.1002/pro.5123

Figure Lengend Snippet: New cellular model of HCU. (a) Growth curves of HEK293A CBS KO cells stably expressing the designated CBS variant. Doubling times determined from the growth curves are summarized in the table on the right. (b) Cell proliferation rate as determined by the BrdU assay. (c) Cell viability assay as determined by the MTT assay. (d) CBS activity assay (10 μg/assay) of clarified cell lysates in the absence and presence of 200 μM SAM. Significant differences ( p < 0.05) compared to CBS WT are noted by asterisk (*) or hashtag (#) in case of CBS activity in the presence of SAM.

Article Snippet: Amount of genomic DNA in cells seeded at ~80% confluency was quantified after 24 h incubation in 5% CO 2 atmosphere at 37°C by using the colorimetric Cell proliferation BrdU ELISA kit (EMD Millipore) according to manufacturer's protocol.

Techniques: Stable Transfection, Expressing, Variant Assay, BrdU Staining, Viability Assay, MTT Assay, Activity Assay