Immunofluorescence. Embryos were dissected in phosphate buffer saline (PBS) with 0.4% bovine serum albumin (BSA), and fixed in 4% paraformaldehyde (PFA) at 4°C for 1 hour. The fix was removed by washing in PBS at 4°C for 2 hours before being placed in 30% sucrose at 4°C overnight. Embryos were washed in OCT at least twice prior to being embedded in OCT. Frozen embryos were sectioned at 10-μm thickness. Slides were washed in antibody solution (10% heat-inactivated goat serum and 0.1% Triton X-100 in PBS) at RT for 10 minutes, and incubated with primary antibodies at 4°C overnight. After washing several times in antibody solution at RT, slides were incubated with secondary antibodies for 1 hour at RT in dark. Slides were washed, and mounted with prolong antifade mounting solution. Primary antibodies used were rabbit anti-Arl13b (serum 503, 1:1500), mouse anti-Cre (Sigma C7988, 1:500), rabbit anti-Olig2 (Millipore AB9610, 1:300), PDGFRα (GeneTex 558774, 1:100), and rabbit anti-Smoothened (Dr. Xxxxxxx Xxxxxxxx, 1:500). Mouse anti-FoxA2, HB9, Nkx2.2, Nkx6.1, Pax6, and Shh primary antibodies were obtained from Developmental Studies Hybridoma Bank, and the 1:10 dilution was used. Secondary antibodies were obtained from Invitrogen for AlexFlour series of anti-rabbit (A11034 for 488nm; A11011 for 568 nm), anti-mouse (A11029 for 488nm; A11031 for 568 nm), and anti-rat (A21471 for 594 nm). The dilution for secondary antibodies was 1:200. For staining mouse embryonic fibroblasts, cells were washed with PBS and fixed in 4% PFA for 10 minutes and then stained as described for embryonic frozen sections. For whole mount staining, E8.5 embryos were fixed in 2% PFA for 20 minutes at RT and washed with PBS for 10 minutes. Embryos were then permeabilized in PBS with 0.1% Triton X-100 and 100mM glycine for 10 minutes, and washed in PBS for 10 minutes. After blocking in blocking buffer (10% calf serum, 0.1% BSA, and 1.5% heat- inactivated sheep serum (HISS) in TBST containing 20mM Tris, 150mM NaCl, 0.05%Tween-20) at RT for 3 hours, embryos were incubated with primary antibody in TBST with 0.1% BSA and 1.5% HISS at 4°C overnight. The next day, embryos were washed in TBST and then incubated with secondary antibody in TBST at RT for 3 hours in dark. Embryos were washed in TBST several times and taken pictures by Leica MZFLIII stereomicroscope.
Immunofluorescence. All immunofluorescent staining experiments were performed on cells seeded at appropriate density on fibronectin coated coverslips. On the day of immunofluorescent labelling, cells were washed 3 times with PBS and fixed with 4% PFA for 20 min. Following another 3 PBS washes, cells were permeabilised with 0.2% X-triton to break the cell membranes for 5 minutes. Following another 3 washes, coverslips were blocked with 5% FBS for 30 min and 3% BSA:PBS for another 30 min. Cells were then incubated with the primary antibody appropriately diluted in 3% BSA:PBS for 2 hours in the dark (table 2-3). Once again, cells were washed 3 times with PBS. Secondary antibodies along with Phalloidin and DAPI were diluted appropriately in 3% BSA:PBS. Cells were then incubated with the prepared mixture for 1h in the dark (table 2-4). Coverslips were then washed twice in PBS and once in ddH2O before being mounted on glass slides with the Fluorsave reagent. Coverslips were left in room temperature overnight and then stored at 4°C in the dark. Cells were imaged on an Olympus IX71 microscope using Image ProPlus AMS software.
Immunofluorescence. For SIRT2 localization experiments, U2OS cells were transfected with SIRT2-FLAG WT or mutants, treated 72 hours post transfection with 5 nM Leptomycin B (Sigma) for 4 hours, fixed in 4% PFA for 10 minutes, and permeabilized in 0.5% triton X-100 for 10 minutes. Cells were blocked in 5% BSA and immunostained with anti-FLAG (Cell Signaling: 2368P) and Alexa Fluor 555 anti-rabbit secondary antibody (Invitrogen) followed by DAPI stain (Southern Biotech). For GFP-ATRIP experiments, a stably transfected GFP-ATRIP U2OS cell line was used. Cells were transfected with nontargeting siRNA (siNS) or siSIRT2-5 followed by transfection with pcDNA3.1 empty vector (EV) or SIRT2-FLAG WT or mutants. SIRT2-FLAG constructs contained wobble mutations to protect exogenous expression from knockdown. Cells were treated with 3mM HU for 24 hours prior to fixation and were fixed and blocked as described above. Cells were immunostained with anti-FLAG (Cell Signaling: 2368P) followed by Alexa Fluor 555 secondary antibody (red), and DAPI Stain. GFP-ATRIP did not require antibody (green). SIRT2-FLAG positive cells were used for quantitation of these experiments with an exception for EV transfected conditions. For spontaneous γH2AX foci experiments, we utilized SIRT2 WT or KO U2OS cells transfected with or without SIRT2-FLAG WT or mutants [95]. Cells were fixed and blocked as described above, immunostained with anti-FLAG (Cell Signaling: 2368P) and anti-γH2AX (Millipore 05- 636) followed by Alexa Fluor 488/555 secondary antibodies and DAPI stain. SIRT2- FLAG positive cells were used for quantitation of these experiments with an exception for non-SIRT2 transfected conditions. Percentage of cells positive for GFP-ATRIP or γH2AX foci was counted from three replicas of 100 cells each. Micronuclei experiments were fixed and processed as described above. Only SIRT2-FLAG positive cells were used in quantitation analysis. The percentage of cells positive for micronuclei was counted from three replicas with 100 cells each. All Images were captured on a Zeiss Observer Z1 microscope equipped with Axio vision Rel 4.8 software. Immunoblot Harvested cells were lysed for 30 minutes on ice in Nonidet P-40 buffer (200 mM NaCl, 1% Nonidet P-40, 50 mM Tris·HCl pH 8.0) freshly supplemented with protease inhibitors. Protein samples were resolved by SDS/PAGE and probed with indicated antibodies: SIRT2 (Santa Xxxx sc-20966), GAPDH (Santa Xxxx sc-25778 or sc-47724), Flag (Sigma F4042), CDK9 (Santa Xxxx sc-13130)...
Immunofluorescence. Wildtype or SIRT2 knockout U2OS cells were transfected with SIRT2-FLAG or SIRT2- FLAG-H187Y expression vectors, or were non-transfected. After 72 h to allow for protein expression, cells were exposed to 0 or 10Gy IR followed by 30 min, 60 min, or 90 min recovery time. Cells were fixed on coverslips with 4% PFA for 10 min, and permeabilized in 0.5% triton X-100 for 10 min. For non-rescue experiments, cells were blocked in 5% BSA and immunostained with anti-γH2AX (Millipore 05-636) and DNA- PKcs phosphoserine 2056 (Abcam, ab18192) followed by Alexa Fluor 555/488 secondary antibodies and DAPI. For rescue experiments, cells were blocked in 5% BSA and immunostained stained with anti-FLAG (Cell Signaling: 2368P) and DNA-PKcs phosphoserine 2056 (Abcam, ab18192) followed by Alexa Fluor 555/488 secondary antibodies and DAPI stain (Southern Biotech). The percentage of cells positive for DNA- PKcs phosphoserine 2056 foci (5 or more foci per nucleus) was counted from 3 biological replicates (100 cells per replica) for each condition.
