Fig 2: ZnPP fluorescent labeling of cellular extracts. (A) Titration of complexes with ZnPP. Cellular lysates as described in Methods were prepared from log phase Huh7 cells and incubated 1 h at RT with various concentrations of ZnPP. Mixtures were electrophoresed on non‐denaturing 0.8% agarose gels and visualized fluorescently using red excitation 608–632 nm and emission 675–720 nm (upper panel) or broad band UV light (lower panel). (B) ZnPP labeling of extracts. ZnPP (10 µM) was added to BSA, non‐specific rabbit IgG, or cellular lysates from Hek293 and Huh7 at the indicated concentrations and incubated for 1 h at RT. Aliquots were then electrophoresed on 0.8% agarose gels and visualized under broad band UV light. (C) Labeling complexes with ZnPP in intact cells. ZnPP (0, 5, and 10 µM) was incubated with Hek293 and Huh7 cultured cells for 24 h, then enzymatic extracts were prepared, electrophoresed on 0.8% agarose gels, and visualized under broad band UV light. (D) ZnPP labeled complexes have mobility shift with anti‐TERT antibodies. Enzymatic cellular extracts (10 µg as protein) were prepared and labeled with 10 µM ZnPP for 1 h at RT. The indicated antibodies were then added, and mixtures incubated at 4°C for 1 h prior to electrophoresis on 0.8% agarose gels and broad band UV light visualization. (ab = antibody). Arrow = mobility shift of anti‐TERT antibodies. (E) Immunostaining of ZnPP labeled complexes blotted to nitrocellulose. Lysates from Huh7 cells were incubated for 1 h at RT with 10 µM ZnPP or vehicle control, then electrophoresed on agarose gels and diffusion‐blotted onto nitrocellulose (NC). ZnPP complexes were visualized in gels by broad band UV and on NC blots by red fluorescence (wavelengths noted above). NC blots were then stained with specific anti‐TERT or anti‐dyskerin antibodies

Fig 3: Fluorescence co‐localization of ZnPP with telomeres and TERT. (A) Co‐localization of ZnPP and telomeres. Synchronized S phase Huh7 cells (left panels) and metaphase chromosomal spreads (right panels) were prepared as described in Methods. Fixed preparations were reacted with the specific fluorescent telomere probe TelC‐Alexa488 and 10 µM ZnPP. Confocal microscopy used Alexa Fluor 488 (green filter) or Alexa Fluor 568 (red filter) to visualize telomeres or ZnPP, respectively, and merged sites (yellow). Yellow arrows (left panels) show co‐localization of telomeres and ZnPP. White arrows, (right panels) show localization of telomeres which do not co‐localize with ZnPP. (B) Co‐localization of ZnPP and TERT in synchronized S phase (left panels) and unsynchronized log phase (right panels). Huh7 cells were incubated with 10 µM ZnPP after labeling with anti‐TERT antibodies. Confocal microscopy was then conducted using Alexa Fluor 488 (green filter, anti‐TERT) or Alexa Fluor 568 (red filter, ZnPP) fluorochromes to visualize TERT or ZnPP, respectively, and merged sites (yellow). Arrows depict example sites of co‐localization of ZnPP and TERT in the nuclei of S phase cells (left panels) or cytoplasm/perinuclear regions of unsynchronized semi‐confluent cells (right panels)

Fig 4: 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 IC50 of telomerase from either source (Table 2). (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 4oC 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)

Fig 5: Effects of MPPs on DNA synthesis, cellular proliferation, and viability. (A) Incorporation of 3H‐thymidine. Early log phase cultures of Huh7 (upper chart) or Hek293 (lower chart) cells were treated with 10 µM of the indicated MPP. The incorporation of 3H‐thymidine was determined at 24, 48, or 72 h of MPP treatment. t‐test, ZnPP versus other MPPs,* p < .001. (B) Inhibition of proliferation. MTT assays were performed after 10 µM MPP treatment of Huh7 cells (upper chart) or Hek293 cells (lower chart) for 0, 1, 2, 3 days. t‐test, ZnPP versus other MPPs, *p < .01, **p < .05. (C) Cell growth and viability. ZnPP (upper chart) and FePP (lower chart) were added to log phase cultures of Huh7 cells. The number of viable cells that excluded trypan blue dye was determined daily using direct cell counting. t‐test 5 and 10 µM (*p < .05), and 20 µM **p < .01 versus other concentrations at days 2 and 3, respectively. (D) Effects of ZnPP in TERT negative (U2OS) as compared to TERT positive (Huh7) cell lines. Early log phase cells were treated with the indicated concentrations of ZnPP for 48 h (upper chart) or BIBR for 72 h (middle chart) and viability then determined using MTT assay. Upper chart, t‐test U2OS versus Huh7 *p < .01. Middle chart, t‐test, U2OS versus Huh7 at 10 and 20 µM *p < .01. For lower chart, vector only plasmid or plasmid pCI neo‐hEST2 containing complete human TERT DNA sequences were transfected into log phase U2OS cells. 24 h later, cultures received the indicated concentrations of ZnPP and then incubated 48 h, followed by MTT assay to determine viability. t‐test, TERT vs vector control cells viability *p < .01. (Right) Immunoblot analysis of TERT in U2OS cells after sham treatment or transfections with plasmid only or human TERT sequences. Untreated Huh7 cell lysate with endogenous TERT is shown as positive control. (E) Apoptosis was measured with BD Biosciences Annexin V FITC assay in Hek293 (upper), Huh7 (middle) and U2OS (lower) panels cells after incubation with the indicated concentration of MPP for 48hr. ANOVA with paired t‐ test, ZnPP apoptosis versus other MPPs,**p < .025, *p < .01. (A‐E) Variability expressed as mean, +/‐ standard deviation, n = 3–6 determinations per point