Ab70369

The rats were sacrificed 7 days after the MCAO model, and the brain tissue was taken for a follow-up study. The rat brain slices attached to the viscous slide were washed with TBS (0.3% Triton X-100 PBS) for 5 min × two times and sealed with blocking solution (0.3% Triton X-100 + 10% PBS of goat serum) at room temperature for 1 h. The rat brain frozen sections were then added with primary antibody PCNA(1:100, Abcam, ab29), Nestin(1:100, Abcam, ab6142), Ki-67(1:100, Cell Signaling Technology, #9129), CUL4A(1:100, Proteintech, 66038-1-Ig), KAP1(1:100, Abcam, ab70369), phospho-KAP1(1:100, Abcam, ab70369), and incubated at 4 °C in a wet box overnight. The following day, wash the brain slides with TBS for 5 min × five times and incubate with goat anti-mouse IgG (H + L)(1:1000, Cell Signaling Technology, #4410) or goat anti-rabbit IgG (H + L)(1:1000, Abclonal, AS007)secondary antibody in a wet box at room temperature for 1–2 h. Afterward, wash the brain slides with TBS for 5 min × five times, and then DAPI staining was added to the brain slides, incubating for 10 min. Finally, wash the brain slides with TBS for 5 min × five times, seal the slide with a sealing tablet, and photograph under Olympus IX73 inverted fluorescence microscope.

MCF7 and MDA-MB-231 breast cancer cell lines (ATCC; STR authenticated) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 100 Units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine (PSG, Sigma-Aldrich) and 10% heat inactivated fetal bovine serum (FBS, First Link). MCF7/LMTK3 and MDA-MB-231/LMTK3, stably over-expressing LMTK3, were cultured in DMEM supplemented with 10% FCS, G418 (500 μg/ml; Invitrogen) and 1% PSG. All cells were incubated at 37 °C in humidified 5% CO₂, and were frequently tested for mycoplasma contamination. Antibodies used: LMTK3 (sc-100418, Santa Cruz Biotechnology); LMTK3 (H00114783-M02, Abnova); β-actin (ab627, Abcam); phospho-histone H2A.X Ser139 (2577, Cell Signaling Technology); phospho-histone H2A.X Ser139 (9718, Cell Signaling Technology); ATM (sc-23921, Santa Cruz Biotechnology); phospho-ATM Ser1981 (4526, Cell Signaling Technology); KAP1 (ab10484, Abcam); phospho-KAP1 Ser824 (ab70369, Abcam). Doxorubicin hydrochloride (D1515) was purchased from Sigma-Aldrich.

Lungs were collected at a treatment endpoint predefined as 27 weeks of age. For tissue preparation, lungs were fixed in 10% phosphate-buffered formaldehyde for 24–48 hrs, dehydrated by incubation in ethanol followed by xylene (70% ethanol, 3 hr; 95% ethanol, 2 hr; 100% ethanol, 2 hr; ethanol-xylene, 1hr; xylene, 3hr) then embedded in paraffin. Paraffin blocks were cut into 5 μm sections, mounted on microscope slides, and stored at room temperature until used. Prepared specimens were stained with hematoxylin and eosin (H&E) (Sigma-Aldrich, St. Louis, MO) and analyzed by immunohistochemistry (IHC) or immunofluorescence using standard protocols. Antibodies used in IHC were to Ki-67 (DAKO, Carpinteria, CA), cleaved caspase 3 (#9664L, Cell Signaling, Beverly, MA), pS⁸²⁴-KAP1 (ab70369, Abcam, Cambridge, MA), pT²⁰²/Y²⁰⁴-ERK1/2 (#4996S Cell Signaling, Beverly, MA) and HSP90 (NB120-2928, Novus Biologicals, Littleton, CO). For immunofluorescence, we used antibody to vimentin (ab92547, Abcam, Cambridge, MA) as primary reagent and secondary Alexafluor 568 tagged donkey anti-rabbit antibody (Life Technologies, Eugene, OR), and counterstained with a 2 μmol/L 4′, 6-diamidino-2-phenylindole (DAPI) (#1652731, Life Technologies, Eugene, OR) solution to visualize DNA.