Journal: Cells

Article Title: Natural Product Library Screens Identify Sanguinarine Chloride as a Potent Inhibitor of Telomerase Expression and Activity

doi: 10.3390/cells11091485

Figure Lengend Snippet: Screening of natural hTERT inhibitors and their verification in the endogenous hTERT-P2A-GFP knock-in HEK293Treporter cell line. ( A ) Schematics of the endogenous hTERT-P2A-GFP HEK293T reporter cell line construction and the screening strategy of a natural product pool for telomerase modulators. Two rounds of screening were carried out and compounds with GFP/RFP ratio ≤ 0.6 were selected as candidates. ( B ) Results from the second round of compound screening, with two candidates highlighted as potential inhibitors. The orange triangle indicates SC while the green rectangle indicates Braz. ( C , D ) Mean fluorescence intensity (MFI) quantification of endogenous hTERT-GFP ( C ) and internal reference dsRed2 ( D ) after 1 μM SC treatment for 48 h. ( E ) hTERT mRNA level by quantitative real-time PCR of reporter cell line upon the treatment of SC (1 μM) for 24 h. DMSO served as the control group. ( F ) Real-time quantitative telomeric repeat amplification protocol (Q-TRAP) assay in reporter cells treated with SC (1 μM). DMSO served as the control group. The screening was performed three independent times. Real-time PCR and Q-TRAP assays are from triplicate samples (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The HEK293T cell line and cancer cell lines, including HTC75, HeLa, DLD1, MDA-MB-231, Hs578t and A549, were routinely cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning; New York, NY, USA) supplemented with 10% Fetal bovine serum (FBS; Excell Bio; Jiangsu, China).

Techniques: Knock-In, Fluorescence, Real-time Polymerase Chain Reaction, Control, Amplification, TRAP Assay

Journal: Cells

Article Title: Natural Product Library Screens Identify Sanguinarine Chloride as a Potent Inhibitor of Telomerase Expression and Activity

doi: 10.3390/cells11091485

Figure Lengend Snippet: SC directly suppressed telomeric repeat extension in vitro without inducing a G-quadruplex motif. ( A ) Schematic representation of immunoprecipitation-based TRAP experiment. FLAG-hTERT-overexpressing HEK293T cells were subjected to immunoprecipitation. The elutes mixed with SC were used to perform the TRAP assay in vitro. The inhibition of natural telomerase activity by SC was presented in a dose-dependent manner, and the fitting curve showed that its IC50 to natural telomerase in vitro was 1.4 μM. ( B ) Purified GST-opTERT was incubated with in vitro-transcribed hTERC for 30 min, then mixed with SC to perform the TRAP assay. SC directly suppressed the activity of reconstituted telomerase at the nanomole level in vitro. ( C ) MST analysis of the interaction of the telomeric G-quadruplex with SC. The EC50 was 120 μM. ( D ) Detection of 2 μM Thioflavin T (ThT) fluorescence intensity at 491 nm for the whole genomic DNA in a K + Tris-HCl buffer. Pyridostatin (PDS) was the positive compound used to induce G-quadruplex structure. ( E ) Representative immunofluorescence images of the G-quadruplex (recognized by BG4 antibody, red) and TRF2 (green) foci in HTC75 cells treated with SC or DMSO for 48 h. Quantification of the number of G-quadruplex foci (recognized by BG4 antibody) per nucleus in compound-treated HTC75 cells (right upper) and quantification of the number of colocalized G-quadruplex foci (recognized by BG4 antibody) and TRF2 in the nucleus (right bottom). In total, 100 nuclei were counted and statistically analyzed. ( F ) A fluorescence polarization binding assay with His-tagged hTRBD and SC was performed, and the EC50 was 24.84 μM; the BSA protein served as a negative control. All the analyses were performed on triplicate samples (* p < 0.05, **** p < 0.0001, ns means no significance).

Article Snippet: The HEK293T cell line and cancer cell lines, including HTC75, HeLa, DLD1, MDA-MB-231, Hs578t and A549, were routinely cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning; New York, NY, USA) supplemented with 10% Fetal bovine serum (FBS; Excell Bio; Jiangsu, China).

Techniques: In Vitro, Immunoprecipitation, TRAP Assay, Inhibition, Activity Assay, Purification, Incubation, Fluorescence, Immunofluorescence, Binding Assay, Negative Control