Ab108997

SKM-1cells were treated with VEM (20 µM), or Bortezomib (BOR, 10 nM), for 48 h. Meanwhile, VEM, at a concentration of 5 µM, or BOR, at a concentration of 4 nM, was added to 1 million MOLM-13 cells for 48 h. AML cell lines were collected and washed three times with phosphate buffer saline (PBS), then blocked in 4% paraformaldehyde, and 0.1% Triton X-100 at room temperature for 1 h. Then AML cell lines were incubated with primary antibody overnight at 4 °C. Primary antibodies were used: TRF2 (Rabbit, Abcam-ab108997), Lamin B1 (rabbit, Abcam-ab133741). AML cell lines were then washed three times in PBS and incubated with secondary antibodies for 1 h at room temperature. The following secondary antibodies were applied in our study: FITC Goat Anti-Rabbit IgG (AS011). Finally, AML cell lines were washed three times (each time for 5 min) with PBS and then incubated with CrystalMount with DAPI for 10 min. Fluorescent images were captured by using a Leica confocal microscope (Leica TCS SP8 SR).

For promyelocytic leukemia protein (PML) and TRF2 co-localization, interphase nuclei were used. The cells were fixed with 3.7% formaldehyde in PBS for 20 min in 1.5 mL tube. After 3 times washing with PBS, the cells were transferred on Polysine Slides (Thermo Scientific™ Shandon™ Polysine Slides, Thermo Scientific, Waltham, MA, USA) and permeabilized with 0.3% Triton X-100 in PBS for 5 min at RT and next blocked with 1% BSA in PBS-T (PBS-Tween20) at RT for 30 min. After washing with PBS-T, the cells were incubated with antibodies: anti-PML (1:200, ab96051) and anti-TRF2 (1:100, ab108997) (Abcam, Cambridge, UK) overnight at 4 °C. After incubation, cells were washed twice with PBS and incubated with secondary antibodies: FITC-conjugated and TexasRed-conjugated, respectively (all at 1:000, F2761, T6390) (Life Technologies, Carlsbad, CA, USA) for 1 h at RT in the dark. Cells were then washed three times with PBS and nuclei were stained with DAPI. Images were taken using an Olympus BX61 fluorescent microscope (Shinjuku, Japan) with objective 20×. To analyze co-localization PML/TRF2, ImageJ software http://rsbweb.nih.gov/ij/ with JACoP plugin was used [16 (link)]. The Pearson’s coefficient was used to calculating a set of co-localization indicators.

Cells were lyzed using RIPA buffer (#P0013C, Beyotime, Shanghai, China) supplemented with Protease and Phosphatase Inhibitors (#A32959, Thermo). Proteins were transferred to PVDF membranes after being separated in SDS/PAGE. The primary antibodies were incubated overnight at 4 °C, and the HRP-conjugated secondary antibodies were incubated for 1–2 h at room temperature. Final detection was performed by using ECL Plus Western Blotting Detection Reagents (#WBULS0500, Millipore). Antibodies against TAZ (#4883), β-catenin (#8480), p-ATR (#2853), p-ATM (#5883), p-BRCA1 (#9009), p-CHK1 (#2348), p-CHK2 (Thr1079, #8654) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against p21 (#sc-6246), hTERT (#sc-393013), Rad51C (#sc-56214) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against TRF1 (#ab129177), TRF2 (#ab108997), POT1 (#ab124784), TIN2 (#ab197894), RAP1 (#ab175329) were obtained from Abcam (Cambridge, MA, USA). Antibodies against GAPDH (#HRP-6004), β-actin (#HRP-60008) were obtained from Proteintech Group Inc. (Wuhan, China).