Anti sp3

ChIP experiments were carried out using a ChIP^TM Enzymatic Assay Kit (Cat#P2083S, Beyotime). Briefly, A549 cells were fixed with 1% formaldehyde and incubated with glycine reagent. Cells were then washed with PBS plus inhibitors of phosphatase and protease, and followed by the preparation of cell nucleus. For DNA fragmentation, the MNase was subsequently used according to the Kit protocols. Following the fracture of nuclear membrane, total extractions were incubated with anti-SP3 (Cat#ab227856, 1:300, Abcam) or anti-IgG isotype (Cat#ab172730, 1:500, Abcam) antibody and Protein A/G Magnetic Beads overnight at 4°C. Using the DNA purification Kit (Cat#D0033, Beyotime) to purify the bead-bound DNA, qPCR was used to detect the enrichment level of the Timeless promoter containing the F2 fragment using specific primer sets shown in S1 Table.

Tissue sections were deparaffinized and Target Retrieval Solution (Dako, Heverlee, Belgium) was used for antigen retrieval. Tissue sections were incubated in the primary antibody, anti-SHIP2 (1:100, Abcam, Cambridge, UK), anti-Sp1 (1:100, Abcam), or anti-Sp3 (1:100, Abcam), overnight at 4 °C. Immunoreactivity was detected using the Dako Envision HRP Detection system/DAB according to the manufacturer’s instructions. IHC staining was evaluated by two independent pathologists using a previously described semi-quantitative scoring system [32 (link)]. Score for staining intensity: 0 (no staining), 1 (light brown), 2 (brown), and 3 (dark brown); for percentage: 0 (≤5%), 1 (6%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (>75%). For statistical analysis, the product score (immunoreactivity score, IRS) of intensity and extent of staining was grouped into low (final score, 0 to 4) or high (final score, 5 to 12).