pbs u3 terc 500  (Addgene inc)

Journal: Pharmacology Research & Perspectives

Article Title: Zinc protoporphyrin binding to telomerase complexes and inhibition of telomerase activity *

doi: 10.1002/prp2.882

Figure Lengend Snippet: MPP inhibition of telomerase enzymatic activity. (A) Telomerase inhibition by MPP in cultured cells. Log phase Huh7, Hek293 and Huh5.15 HCV replicon cells, (upper, middle, and lower panels, respectively), were incubated with MPPs (ZnPP, SnPP, or FePP) for 48 h, then telomerase activity was determined by TRAP assay in enzymatic lysate. Points represent mean ± SD, n = 6, ANOVA, # p < .01, * p < .001, ** p < .01 paired t‐test of ZnPP versus other MPPs or vehicle only controls. (B‐D) Telomerase inhibition by MPP in enzymatic extracts. Enzymatic extracts were prepared from semi‐confluent phase Huh7 cells and aliquots were assayed in triplicate using TRAP assay. MPPs were added to RT‐PCR reactions either before or after the RT telomere elongation step to test whether MPPs inhibit Taq polymerase. Visualization of amplified telomeric products was on 10% non‐denaturing polyacrylamide gels using SYBR fluorescence labeling (upper panels) followed by quantification with densitometry and plotted (lower panel). IC = Internal Taq polymerase control. Plotted points represent the mean ± SD with n = 3. (E) MPP inhibition of telomerase activity as determined with direct α‐ 32 P‐dGTP extension assays. Aliquots of IP eluates from 10 7 Hek293 cell pellet lysates overexpressing hTERT, TERC, and dyskerin were incorporated into direct telomere extension assays using biotin‐linked DNA substrate. Reactions were incubated at 37ºC for two hours, then biotin labeled products purified with strepavidin linked agarose beads and electrophoresed on denaturing acrylamide gels. Bands were visualized radiographically (upper panel) and relative activity was quantified with densitometry (lower panel). Points represent mean ± SD, n = 3, ANOVA * p < .01 paired t test of ZnPP versus other MPPs

Article Snippet: Telomerase RNA component (TERC) plasmid (pBS U3‐hTR‐500) was also obtained from Addgene ( plasmid # 28170), a gift from Dr. Kathleen Collins.,

Techniques: Inhibition, Activity Assay, Cell Culture, Incubation, TRAP Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Fluorescence, Labeling, Control, Purification

Journal: Pharmacology Research & Perspectives

Article Title: Zinc protoporphyrin binding to telomerase complexes and inhibition of telomerase activity *

doi: 10.1002/prp2.882

Figure Lengend Snippet: Inhibition of telomerase by MPPs in direct telomere extension assays

Article Snippet: Telomerase RNA component (TERC) plasmid (pBS U3‐hTR‐500) was also obtained from Addgene ( plasmid # 28170), a gift from Dr. Kathleen Collins.,

Techniques: Inhibition, Telomerase Assay

Journal: Pharmacology Research & Perspectives

Article Title: Zinc protoporphyrin binding to telomerase complexes and inhibition of telomerase activity *

doi: 10.1002/prp2.882

Figure Lengend Snippet: ZnPP inhibition and labeling of Immunoprecipitated TERT complexes. (A) ZnPP binds TERT IP complexes. Left panel : Immunoprecipitation was performed on lysates from Hek293 cells transfected with TERT and TERC plasmid constructs as described in the Methods. The IP were then re‐suspended and incubated with ZnPP (10 µM) for 1 h at RT prior to electrophoresis on 0.8% agarose gels and visualized under UV light. Right panel : Identical aliquots were also denatured for SDS‐PAGE and evaluated on WB. (B) ZnPP inhibition of telomerase activity in cellular lysates and IP. TRAP assays were performed with IP or crude cellular lysates in the presence of various concentrations of ZnPP. No statistical difference of ZnPP IC 50 of telomerase from either source (Table ). (C) Nuclease digestion of ZnPP labeled complexes. Upper panel : Enzymatic extracts or IP (10 µg protein) were treated with DNase 1 or RNase A (0.1 mg/ml at 4 o C for 1 h), then labeled with ZnPP (10 µM) for 1 h at RT, before electrophoresis on 0.8% agarose gels and visualization of ZnPP by red fluorescence emission. Lower panel : Following electrophoresis, native proteins were transferred to NC by diffusion blotting and stained with anti‐TERT antibodies. For whole cell lysates, GAPDH was used as a loading control after lysates were electrophoresed on SDS‐denaturing polyacrylamide gels and stained using WB methods (LB = lysis buffer); (IP= immunoprecipitate); (N = Native)

Article Snippet: Telomerase RNA component (TERC) plasmid (pBS U3‐hTR‐500) was also obtained from Addgene ( plasmid # 28170), a gift from Dr. Kathleen Collins.,

Techniques: Inhibition, Labeling, Immunoprecipitation, Transfection, Plasmid Preparation, Construct, Incubation, Electrophoresis, SDS Page, Activity Assay, Fluorescence, Diffusion-based Assay, Staining, Control, Lysis