Telomere assays
Immunofluorescence and fluorescence in situ hybridization (IF-FISH): Cells
cultured on coverslips were treated with Bazhen decoction (control, low
dose (10μg/mL), high dose (20μg/mL)) for 48 hours. After washing three
times in 1×PBS, cells were fixed for 10 minutes in 2% sucrose and 2%
paraformaldehyde, and permeabilized with 0.5% NP-40. After blocking in
blocking solution (2% Gelatin and 0.5% BSA in 1×PBS), the primary
antibody were applied overnight at 4°C and followed by secondary
antibody incubation for 1h at room temperature in the dark. After
washing three times in 1×PBST, the cells were fixed with 4%
paraformaldehyde for 10 minutes at room temperature, and hybridized with
a telomere PNA-FISH probe 5′-FITC-green-(TTAGGG)-3′ (Panagene).
Coverslips were washed and counterstained with anti-fade mounting medium
with DAPI (Solarbio). The primary antibodies for IF-FISH: anti-Recql4
(1:200, Invitrogen), anti-Blm (1:200, Invitrogen).
Telomere DNA qPCR assay: The cultured cells (1x105cells/well in 6-well plate) were treated with Bazhen decoction (control,
low dose (10μg/mL), high dose (20μg/mL)) for 48hr. Then the cells were
passaged for six times (1x105 cells/well in 6-well
plate) and continued with the Bazhen decoction treatment. The
7th passage cells were harvested and the genomic DNA
was purified for Syber green based real time PCR. The telomere DNA
primer sequences were: Forward:
5’-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3’, Reverse:
5’-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3’. The reference 36B4 DNA
primer sequences were: Forward: 5’-ACTGGTCTAGGACCCGAGAAG-3’, Reverse:
5’- TCAATGGTGCCTCTGGAGATT-3’.