Journal: Nucleic Acids Research

Article Title: Human cytomegalovirus regulates host DNA repair machinery for viral genome integrity

doi: 10.1093/nar/gkaf1445

Figure Lengend Snippet: USP1 restricts CMV replication. MRC-5 fibroblasts were reverse-transfected with a siRNA pool targeting UAF1 or USP1 or an NTC. At two days post-transfection, cells were infected with CMV TB40/E-GFP wild-type (WT) or TB40/E-∆ UL138 STOP at an MOI of 1 and cells were harvested at 96 hpi. ( A ) Viral genome copy number was determined by qPCR using a TB40/E BAC standard curve and primer set designed for the region of the viral genome encoding the β2.7 transcript. The graph represents the fold change in viral genome copy number relative to siNTC. ( B ) Virus yields were measured by TCID 50 and normalized relative to the WT siNTC condition. For panels (A) and (B), statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparisons test. Asterisks (** P < .01, *** P < .001, **** P < .0001) represent statistically significant differences determined for four independent experiments. ns, nonsignificant. ( C ) Whole cell lysates were collected at two days post-transfection to determine efficiency of USP1 knockdown by immunoblotting using monoclonal antibodies for USP1 and tubulin as a loading control and secondary antibodies conjugated to DyLight™ 680 (mouse) or 800 (rabbit). Densitometry analysis of three biological replicates reveals an average 68% knockdown compared to siNTC. ( D ) At two days post-transfection, RNA was collected for RT-qPCR to determine the efficiency of UAF1 knockdown relative to siNTC. H6PD was used as a housekeeping control for cell number. Over all experiments, an average knockdown efficiency of 81% was achieved. A paired t- test was used to determine statistical significance, **** P < .0001, over multiple independent experiments.

Article Snippet: Primary human lung MRC-5 fibroblasts (ATCC CCL-171) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM l -alanyl-glutamine, 0.1 mM nonessential amino acids, 100 U/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Transfection, Infection, Virus, Knockdown, Western Blot, Bioprocessing, Control, Quantitative RT-PCR

Journal: Neoplasia (New York, N.Y.)

Article Title: Identification of BET inhibitors (BETi) against solitary fibrous tumor (SFT) through high-throughput screening (HTS)

doi: 10.1016/j.neo.2025.101244

Figure Lengend Snippet: Identification of Mivebresib as an efficient and specific anti-SFT (Solitary Fibrous Tumor) agent. (a) Secondary high-throughput screening (HTS) was performed using the Moffitt-ns and immortalized lung fibroblast cells. The CTG effects of both cell lines under all four doses were plotted. The green line indicated the differences in suppression effects between Moffitt-ns and Lf > 40 %. The blue line indicated that the suppression effects at the highest dose should be > 50 % in Moffitt-ns cells. 3 compounds were shown (Mivebresib: green, Zinc Pyrithione: red, and TAK-901: yellow). (b-f) Confirmatory MTS assays were performed in two primary SFT cells (INT-SFT and IEC139) and two control LMS cells (SKUT-1 and CP0024). Only Mivebresib showed efficient and selective cell proliferation-suppressing effects in SFT cells. (): INT-SFT; (): IEC139; (): SKUT-1; (): CP0024.

Article Snippet: The hTERT-immortalized human lung fibroblast cell line (Lf) was acquired from the American Type Culture Collection (catalog number: CRL-4058) and maintained at 37 °C, 100 % humidity, and 5 % CO 2 .

Techniques: High Throughput Screening Assay, Control