Journal: Oncotarget

Article Title: Elucidating the novel BRCA1 function as a non-genomic metabolic restraint in ER-positive breast cancer cell lines

doi: 10.18632/oncotarget.26093

Figure Lengend Snippet: ( A – D ) Nuclear (N), cytoplasmic (C) and whole cell extracts (T) were prepared from ER-negative breast cancer cells ( A) MDA MB-231 and (B) Hs578T, the ER-positive (C) MCF7 and (D) T47D breast cancer cells, as well as a ( F ) normal mammary epithelial cell line MCF10A. Protein extracts were resolved by SDS-PAGE gel and immunoblotted with antibodies against BRCA1, Lamin A/C and α-tubulin. Lamin A/C and α-tubulin were used to loading controls for the nuclear and cytoplasmic fractions respectively. Representative images of immunofluorescence staining for BRCA1 localisation in ( E ) Hs578T, MDA-MB-231, MCF7 and T47D breast cancer cells and ( G ) normal mammary epithelial cells MCF10A are shown. The cells were labelled with anti-BRCA1 (Ab-1) antibody and the signal was detected with green fluorescent Alexa Fluor ® 488 anti–Mouse IgG. The nuclei were counterstained with blue fluorescent 4′,6-diamidino-2-phenylindole (DAPI). Cells were visualised under Zeiss Axio Vert A1 inverted microscope (Zeiss) and the images were captured with a QIClick CCD Camera (Q Imaging) and processed with Q-capture pro software. Scale bar represents 50 µm.

Article Snippet: Human breast cancer cells, MCF-7, T47D, MDA-MB 231 and Hs578T were purchased from the European Collection of Authenticated Cell Cultures (ECACC) and maintained as previously published [ ].

Techniques: SDS Page, Immunofluorescence, Staining, Inverted Microscopy, Imaging, Software

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

Journal: iScience

Article Title: Conserved role of FOXC1 in TNBC is parallel to FOXA1 in ER+ breast cancer

doi: 10.1016/j.isci.2024.110500

Figure Lengend Snippet: Identification of FOXC1 cofactors (A) Protein co-interactors of FOXC1 identified in Hs578t and BT-549 via RIME in two biological replicates, after subtracting those identified in negative IgG control. (B) Western blot depicting co-immunoprecipitation of FOXC1 using antibody against NR2F2 with input and IgG negative controls.

Article Snippet: Hs578t , AddexBio , C0006016, RRID:CVCL_0332.

Techniques: Control, Western Blot, Immunoprecipitation

Journal: iScience

Article Title: Conserved role of FOXC1 in TNBC is parallel to FOXA1 in ER+ breast cancer

doi: 10.1016/j.isci.2024.110500

Figure Lengend Snippet:

Article Snippet: Hs578t , AddexBio , C0006016, RRID:CVCL_0332.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Sequencing, Generated, Gene Expression, Mass Spectrometry, Knock-Out, Software, Blocking Assay