Ab20669

Biotinylated RNA pulldown assays were performed as described previously.^[7 (link)
^] Briefly, oligonucleotides of lnc‐HZ10 or its various segments were designed, synthesized, and in vitro transcribed from pGEM‐T‐lnc‐HZ10 or pGEM‐T‐lnc‐HZ10‐S1‐5 (Addgene) (sequences in Table S6, Supporting Information). All the transcripts were biotin‐labeled by T7 RNA polymerase using Biotin RNA Labeling Mix (Roche, Basel, Switzerland). After treatment with DNase I, the RNAs were purified with RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The transcripts were then incubated with trophoblast cell lysates at 4 °C overnight. Finally, the protein‐RNA complexes were separated from streptavidin magnetic beads, and the proteins were analyzed by Western blotting with anti‐BRCA1 (1:1000, MS110, Invitrogen) or anti‐γ‐H2AX (1:1000, ab20669, Abcam) antibody.

RIPA buffer (PC104, Epizyme, China) was applied to extract protein from GBM tissues followed by centrifugation. The lysed protein was quantified utilizing BCA kit (ZJ101, Epizyme, China). After electrophoresis (10% SDS-PAGE), the protein was blotted onto nitrocellulose membranes. After being sealed, the membranes were immersed in primary antibodies at 4°C overnight. A further 60 min reaction was done after the addition of secondary antibodies. After being developed with an ECL luminescence reagent (P1000, Applygen, China), the signals were analyzed by a gel imaging system. The primary antibodies of Cyclin D1 (1 : 200, ab16663), C-myc (1 : 1000, ab32072), Bad (1 : 1000, ab32445), p-p53 (1 : 5000, ab33889), ATM (1 : 1000, ab201022), Histone H2A.X (1 : 1000, ab20669), p-Histone H2A.X (1 : 1000, ab81299), phospho-STAT3 (Ser727) (1 : 1000, ab32143), phospho-STAT3 (Tyr705) (1 : 10000, ab267373), STAT3 (1 : 2000, ab68153), and GAPDH (1 : 10000, ab181602) were gained from Abcam (UK). The primary antibodies of p-AKT (1 : 2000, #4060), AKT (1 : 1000, #4685), Cleaved Caspase-9 (1 : 1000, #9507), and p-ATM (1 : 1000, #2851) were obtained from CST (USA). PD-L1 (1 : 1000, abx179111) was bought from Abbexa (USA). Pro-Caspase-9 (1 : 2000, AF6348) was bought from Affinity (USA).

Cells were washed twice with PBS (Thermo Scientific, Waltham, USA), directly solubilized in denaturing sample buffer and then subjected to SDS-PAGE. Proteins were electro-transferred to 0.2 µm nitrocellulose sheets (Bio-Rad, Hercules, USA) for immunodetection with the following primary antibodies: H2A.X (1:5000; ab20669, Abcam, Cambridge, MA, USA); HK2 (1:1000; C64G5, Cell Signaling Technology Inc); Tubulin (1:2000; 11H10, Cell Signaling Technology Inc). Immune complexes were detected with goat coupled anti-rabbit or horse coupled anti-mouse IgG antibodies (Cell Signaling Technology Inc). Cropped images for the western blots and molecular weight are shown in the main and supplementary figures.

Cells were washed twice with PBS, solubilized in denaturing sample buffer and then subjected to SDS-PAGE. Proteins were electrotransferred to 0.2 µm Protran BA 83 nitrocellulose sheets (Invitrogen, Carlsbad, CA, USA) for immunodetection with the following primary antibodies: H2AX (1:2000; ab20669, Abcam, Cambridge, MA, USA); NRF2 (1:2000; MAB3925, R&D system, Minneapolis, MN, USA); NQO1 (1:3000; ab34173, Abcam, Cambridge, MA, USA); GCLC (1:2000; ab53179, Abcam, Cambridge, MA, USA). Immune complexes were detected with horseradish peroxidase coupled anti-rabbit or anti-mouse IgG antibodies (Amersham^TM, GE Healthcare, Pittsburgh, PA, USA).

The cells were fixed for 20 min with freshly prepared 2% paraformaldehyde in phosphate-buffered saline (PBS). After washing with PBS, the cells were permeabilized with pre-chilled ethanol 70% for 20 min. The samples were then incubated for 30 min with 5% bovine serum abumin (BSA) in PBS containing 0.5% Tween-20 and 0.1% Triton X-100 (PBS-TT) for blocking. The cells were incubated for 2 h at room temperature with primary antibodies diluted in 1% BSA–PBS–TT: γ-H2A.X (1:500; 05-636, Millipore); 53BP1 (1:500; NB100-305, Littleton, CO, USA); E-cadherin (1:250; ab76055, Abcam); H2A.X (1:500; ab20669, Abcam); ZEB1 (1:100; sc-25388, Santa Cruz Biotechnology); Slug (1:100; sc-15391, Santa Cruz Biotechnology); and β-catenin (1:500; 610153, BD Biosciences). After washing with PBS–TT, the cells were stained with goat anti-rabbit Alexa fluor488 or goat anti mouse Alexa fluor555 diluted in 1% BSA–PBS–TT for 1 h at room temperature. Finally, the cells were washed in PBS and coverslips were mounted for analysis. Fluorescent images were captured using a confocal microscope (Nikon PCM2000).

HCT116 cells (shCTRL, shH2A.X, parental and H2A.X KO) were processed for ChIP using Magna ChIP A/G kit according to the manufacturer's instructions (Millipore, MA, USA). Briefly, 15 × 10⁶ (ref. 6 (link)) cells were treated with formaldehyde to crosslink chromatin, lysed using buffer provided in the kit. Sheared DNA corresponding to 1 million cells was incubated overnight at 4 °C with primary antibodies for H2A.X (ab20669, Abcam, Cambridge, MA, USA), or H3K9ac (ab4441, Abcam), or H3K27ac (ab4729, Abcam) or normal rabbit IgG (sc-2027, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The chromatin was then washed, the crosslinking was reversed and the DNA was purified using phenol–chloroform precipitation before PCR analysis. The following sets of primers mapping the promoters regions were used: Slug Forward 5′-TCCGAACAAACCCTCACATAG-3′; Slug reverse 5′-CACACAAACTGGAACCTGGA-3′; ZEB1 Forward 5′-GGCGCAATAACGGTGAGT-3′; ZEB1 reverse 5′-ACTTTCCCACTCCACTTTGC-3′; GAPDH forward 5-TACTAGCGGTTTTACGGGCG-3′; GAPDH reverse 5′-GAGGCTGCGGGCTCAATTT-3′.