cell lines  (ATCC)

Journal: Scientific Reports

Article Title: The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants

doi: 10.1038/s41598-021-87344-2

Figure Lengend Snippet: Analysis of the cytotoxic effects of berberine, coptisine, and palmatine. ( a ) PFGE analysis of DSB accumulation after treatment with aphidicolin combined with extracts of Phellodendron Bark and Coptis Rhizome . ( b ) PFGE analysis of DSB accumulation after treatment with aphidicolin combined with extracts of berberine and coptisine. ( c ) PFGE analysis of DSB accumulation after treatment with Z-VAD-FMK combined with extracts of berberine and coptisine. ( d ) Time-course PFGE analysis of DSB accumulation after treatment with CPT, palmatine, berberine and coptisine. ( e ) Quantification of broken DNA. The data are presented as fold inductions relative to the untreated control. ( f ) Survival curves of MRC5sv cells against berberine, coptisine, palmatine, and magnoflorine. ( g ) Survival curves of MRC5sv cells treated with CPT and etoposide.

Article Snippet: Using CPT-resistant cell lines (CPT-K5 and CEM-C2), berberine and coptisine were titrated in the presence of 1 μM CPT, which is higher than the 90% viability concentration on these cell lines, and compared the results with those obtained in the absence of CPT.

Techniques: Control

Journal: Scientific Reports

Article Title: The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants

doi: 10.1038/s41598-021-87344-2

Figure Lengend Snippet: Analysis of Topo I inhibition by BIAs in vivo. ( a ) Western blot analysis of Topo I after transfection with siRNA against the Top1 gene. ( b ) PFGE analysis of DSB accumulation after treatment with transient Topo I depletion by siRNA combined with berberine and coptisine. ( c ) Quantification of broken DNA per total DNA after treatment with transient Topo I depletion by siRNA combined with berberine and coptisine. The means and SEs were determined from four independent experiments. ( d ) Analysis of the accumulation of the Topo I-DNA complex by ICE assay. Following the manufacturer’s recommended protocol, the cells were treated with 50 μM CPT, coptisine, and berberine, and the Topo I-DNA complexes were visualized by immunoblotting using an anti-Topo I antibody.

Article Snippet: Using CPT-resistant cell lines (CPT-K5 and CEM-C2), berberine and coptisine were titrated in the presence of 1 μM CPT, which is higher than the 90% viability concentration on these cell lines, and compared the results with those obtained in the absence of CPT.

Techniques: Inhibition, In Vivo, Western Blot, Transfection

Journal: Scientific Reports

Article Title: The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants

doi: 10.1038/s41598-021-87344-2

Figure Lengend Snippet: Analysis of Topo I inhibition by BIAs in vitro. ( a,b ) Inhibition of the relaxation activity of Topo I by berberine, coptisine, and palmatine. ( c ) Relaxation activity of Topo I after treatment with magnoflorine. ( d ) Detection of nicked DNA based on the effects on the relaxation activity of Topo I by CPT, coptisine (Cop), berberine (Ber), and palmatine (Palm). ( d ) NMR titration curves, 1 H chemical shift changes versus the concentration of dsDNA, and dissociation constant (Kd) values for coptisine, berberine, and palmatine. The inset shows the proton in the alkaloid backbone used for the NMR analysis.

Article Snippet: Using CPT-resistant cell lines (CPT-K5 and CEM-C2), berberine and coptisine were titrated in the presence of 1 μM CPT, which is higher than the 90% viability concentration on these cell lines, and compared the results with those obtained in the absence of CPT.

Techniques: Inhibition, In Vitro, Activity Assay, Titration, Concentration Assay

Journal: Scientific Reports

Article Title: The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants

doi: 10.1038/s41598-021-87344-2

Figure Lengend Snippet: Analysis of the inhibitory effects of BIAs in nicking and rejoining assays. ( a ) Schematic representation of the nicking assay. Nicked DNA products were detected by the Cy-3 fluorescent signal. Unaffected DNA molecules were detected as 36-mer fragments, and nicked DNAs were detected as 22-mer fragments. ( b ) Inhibitory effects of treatment with CPT, berberine (Ber), and coptisine (Cop) on Topo I-dependent nicking activity. ( c ) Schematic representation of the rejoining assay. The rejoined products were detected by Cy-3 and Cy-5 fluorescent signals. Unaffected DNA molecules were detected as 18-mer fragments on a Cy-3-visualized gel, and nicked DNAs were detected as 36-mer fragments on both the Cy-3- and Cy-5-visualized gels. ( d ) Inhibitory effects of treatment with CPT, berberine (Ber), and coptisine (Cop) on Topo I-dependent re-joining activity.

Article Snippet: Using CPT-resistant cell lines (CPT-K5 and CEM-C2), berberine and coptisine were titrated in the presence of 1 μM CPT, which is higher than the 90% viability concentration on these cell lines, and compared the results with those obtained in the absence of CPT.

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants

doi: 10.1038/s41598-021-87344-2

Figure Lengend Snippet: Analysis of the inhibitory effects of BIAs against CPT-resistant Topo I proteins. ( a ) Schematic representation of CPT-resistant TOP1 mutations. ( b ) Relaxation activity of CPT-resistant Topo I proteins in the presence of CPT in vitro. ( c ) Inhibitory effect of coptisine, berberine, epiberberine and palmatine on CPT-resistant Topo I proteins in vitro.

Article Snippet: Using CPT-resistant cell lines (CPT-K5 and CEM-C2), berberine and coptisine were titrated in the presence of 1 μM CPT, which is higher than the 90% viability concentration on these cell lines, and compared the results with those obtained in the absence of CPT.

Techniques: Activity Assay, In Vitro

Journal: Scientific Reports

Article Title: The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants

doi: 10.1038/s41598-021-87344-2

Figure Lengend Snippet: Analysis of the inhibitory effects of BIAs against CPT-resistant cells and Topo I proteins. ( a ) Analysis of CPT resistance of CPT-K5 cells carrying D533G mutations in the TOP1 gene. ( b,c ) Survival curve of RPMI-8402 and CPT-K5 cells after treatment with berberine ( b ) and coptisine ( c ). ( d ) Analysis of CPT resistance of CEM/C2 cells carrying N722S mutations in the TOP1 gene. ( e,f ) Survival curve of CCRF-CEM and CEM/C2 cells after treatment with berberine ( e ) and coptisine ( f ).

Article Snippet: Using CPT-resistant cell lines (CPT-K5 and CEM-C2), berberine and coptisine were titrated in the presence of 1 μM CPT, which is higher than the 90% viability concentration on these cell lines, and compared the results with those obtained in the absence of CPT.

Techniques:

Journal: Scientific Reports

Article Title: The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants

doi: 10.1038/s41598-021-87344-2

Figure Lengend Snippet: Effect of treatment with CPT combined with berberine and coptisine. ( a ) Effect of treatment with CPT combined with berberine and coptisine on the relaxation assay in vitro. ( b-e ) Effect of treatment with CPT combined with berberine and coptisine on the survival of CPT-resistant cell lines, as determined through the MTT assay. ( b,c ) Effect of treatment with CPT combined with berberine ( b ) and coptisine ( c ) on the survival of the CPT-K5 cell line, as determined through the MTT assay. ( d,e ) Effect of treatment with CPT combined with berberine ( d ) and coptisine ( e ) on the survival of CEM/C2 cells, as determined through the MTT assay.

Article Snippet: Using CPT-resistant cell lines (CPT-K5 and CEM-C2), berberine and coptisine were titrated in the presence of 1 μM CPT, which is higher than the 90% viability concentration on these cell lines, and compared the results with those obtained in the absence of CPT.

Techniques: In Vitro, MTT Assay