Ab5079

Immunohistochemistry for the visualization of neuronal nuclear antigen (NeuN), rat endothelial cells antigen 1 (RECA-1), and Iba-1 positive cells expression was performed. To begin the procedure, six sections on average were selected in each brain region, after being blocked with 10% normal goat and rabbit serum for 1 h. In brief, the sections were incubated overnight at 4 °C with a NeuN antibody (1:1000, ab134014, Abcam, Cambridge, UK), RECA-1 antibody (1:1000, ab9774, Abcam, Cambridge, UK), Iba-1 antibody (1:500; ab5079, Abcam, Abcam, Cambridge, UK), and 4HNE (1:200; HNE11-S, Alpha Diagnostic International, San Antonio, TX, USA). The sections were then incubated with the biotinylated rat and mouse secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA) with 0.3% Triton X-100 in PBS for 2 h at RT and subsequently incubated with antibody–biotin–avidin–peroxidase complex solution (Vector Elite ABC kit^®; Vector Laboratories, Burlingame, CA, USA) for 1 h at RT. Finally, the sections were stained with a 3.3′-diaminobenzidine tetrahydrochloride (DAB kit; Vector Laboratories, Burlingame, CA, USA). The sections were finally mounted onto gelatin-coated slides. The slides were air-dried overnight at room temperature, and the coverslips were mounted using Permount^® (Vector Laboratories, Burlingame, CA, USA).

Fenghui Biotechnology Co., Ltd. (Hunan, China) provided the LINC00955 precursor overexpression plasmid, and MiaoLingBio provided the GFP-Sp1 plasmid (Wuhan, China). PHIP-knockdown plasmids were from Open Biosystem. HA-CDK2, HA-DNMT3B, HA-TRIM25, a set of TRIM25-targeting shRNA plasmids, the PHIP promoter-driven luciferase reporter, the DNMT3B promoter-driven luciferase reporter, and deleted LINC00955 fragments were constructed in the laboratory. Antibodies against Sp1 (9389), HA (3724), FOXO1 (2880), and FOXO3A (12,829) were sourced from CST (Boston, MA, USA). Santa Cruz Biotechnology (Dallas, TX, USA) provided antibodies against cyclin D1 (sc-20044), GFP (sc-9996), CDK2 (sc-6248), CDK4 (sc-260), CDK6 (sc-7961), Sp2 (sc-17814), and Sp3 (sc-28305). Antibodies against β-actin (Ab0011) were purchased from Abways Technology (Shanghai, China). Antibodies against tubulin (Ab7291), KLHL6 (Ab182163), and FOXC1 (Ab5079) were purchased from Abcam (Cambridge, MA, USA). Proteintech (Wuhan, China) sold antibodies against the following: PHIP (20,933–1-AP), TRIM25 (12,573–1-AP), DNMT1 (24,206–1-AP), DNMT3A (20,954–1-AP), and cyclin E2 (11,935–1-AP). Antibodies against DNMT3B (52A1018) were purchased from Novus Biologicals (USA).

ChIP was performed as previously described for U2OS cells [6 (link)] and Flag-biotin-tagged GATA4 [5 (link)]. ChIP-sequencing was previously described [8 (link)]. Motif analysis was performed using Meme Suite [25 (link)]. FOXC1 was immunoprecipitated with a goat polyclonal antibody (Abcam, ab5079). The specific primers for SYBR Green assays used for qPCR are listed in Supplemental Table S1. All data were compared to the percent input and a negative control region [8 (link)].