Znf333

Cell lysates were immunoblotted with primary antibodies targeting ALKBH5 (Abcam), ZNF333 (Invitrogen), CDX2 (Abcam), VIL1 (Abcam), KLF4 (Abcam), YTHDF2 (Abcam), p65 (CST), p-p65 (CST), FLAG (CST), and GAPDH (Abcam). The intensity of the fluorescence was detected using a chemiluminescence system (Amersham Biosciences, Piscataway, NJ, USA), and quantified using Quantity One software (Bio-Rad, USA).

The IHC assay was conducted with antibodies against human ALKBH5 (Abcam), ZNF333 (Invitrogen), CYLD (Abcam), CDX2 (Abcam), or p-p65 (CST). The final staining was divided into three grades (negative, weak, and strong) based on the staining intensity and area.²⁷ (link) The staining intensity was scored as 0 (no staining), 1 (weak), or 2 (strong). The staining area was scored as follows: 0 (≤10% positive staining), 1 (11%–25% positive staining), 2 (26%–50% positive staining), 3 (51%–75% positive staining), and 4 (≥75% positive staining). Staining intensity and staining area were summed up to give a final total score index: an overall score of ≤3 was defined as negative expression, of >3–≤6 as weak expression, and of >6 as strong expression. It was also divided into high-expression (strong staining) and low-expression (negative and weak staining) groups.

Cells were fixed in 1% formaldehyde for 20 min and permeabilized with Triton X-100 (0.2%) for 5 min. Next, cells were washed 3 times in PBS, blocked for 1 h with 2% BSA, and incubated with primary antibodies specific for ALKBH5 (Abcam), ZNF333 (Invitrogen), p65 (CST), CDX2 (Abcam), VIL1 (Abcam), and KLF4 (Abcam) for 2 h. They were then washed 3 times in PBS and incubated with secondary antibodies for 1 h at room temperature.