Bx61 fluorescence microscope

HUVECs on round glass slides in 12-well plates were fixed for 30 min. with 4% paraformaldehyde in PBS. After fixation, cells were washed for washed 3X in PBS for 5 min. each before permeabilization with 0.2% Triton X-100 in PBS for 15 min. Cells were washed 3X in PBS for 3 min. each before blocking for 2 hours at room temperature with 5% goat serum in PBS. Cells were then incubated overnight in a humidified chamber at 4°C with anti-HMGB2 (LSBio, Seattle, WA). Slides were washed 3X for 5 min. each in PBS before incubation for 1 hour in the dark at room temperature with the secondary antibody anti-mouse Alexa Fluor 555 (Invitrogen, Carlsbad, CA). Cells were then washed 3X for 5 min. each in 1X PBS before incubation in the dark for 10 min. at room temperature with DAPI at 1:5000 in PBS. Lastly, cells were washed 3X for 3 min. each with ddH₂O before mounting on glass slides with Fluoromount Mounting Medium (Sigma-Aldrich, Darmstadt, Germany). Cells were visualized using an Olympus BX61 fluorescence microscope coupled with a Hamamatsu ORCA-ER camera and using Slide Book 4 software version 4.0.1.44 (Intelligent Innovations, Inc.; Denver, CO). HMGB2 secreting cells were blindly counted by a laboratory member who did not know the conditions and at least 200 cells were counted per coverslip at 40X magnification.

Immunostaining for p53BP1 and TRF2 was performed for HeLaII cells plated
onto glass coverslips and processed for RNAi. After the 48 hour transfection
period, cells were extracted with Tx buffer [0.5% Triton X-100, 20mM Hepes-KOH
pH 7.9, 50mM NaCl, 3mM MgCl₂, 300mM sucrose] for 10 min at RT. After
two PBS washes, the cells were fixed with PBS/3% paraformaldehyde, 2% sucrose
for 10 min at RT. After two PBS washes, cells were permeabilized with Tx buffer
for 10 min at RT, washed twice with PBS and blocked with PBG [PBS/0.2% fish
gelatin. 0.5% BSA] for 30 minutes. Coverslips were then incubated with the
rabbit anti-p53BP1 antibody (Novus NB100-304A-1), at a concentration of 1:500 in
PBG overnight. Cover slips were then rinsed three times with PBG solution and
incubated with secondary TRITC-conjugated goat anti-rabbit antibody (Jackson
Immunoresearch) in PBG at a concentration of 1:500 for 45 min at RT. Cover slips
were rinsed two times with PBG. Coverslips were then incubated with PBG and
4,6-diamidino-2-phenylindole (DAPI) at 100 ng/ml to visualize the nuclei.
Coverslips were mounted onto slides with embedding media. Images were collected
with an Olympus BX61 fluorescence microscope using a 60X objective connected to
a Hamamatsu ORCA-ER CCD camera, controlled by the SlideBook 5.1 image capture
software. The telomere FISH shown in figure S1C was performed as described in (2 (link)).