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. 2022 Aug 17;12(8):e945. doi: 10.1002/ctm2.945

FIGURE 5.

FIGURE 5

FAM135B increases TIP60 histone acetyltransferase (HAT) activity and promotes TIP60 and ATM interactions. (A and B) FAM135B affects the HAT activity of TIP60. KYSE30 cells (A) were transfected with FLAG‐FAM135B or empty vector and MYC‐TIP60. KYSE510 cells (B) were transfected with FAM135B (siFAM135B) or control siRNA (siCTRL) and MYC‐TIP60. Nuclear proteins were extracted from the cells treated earlier, then the protein MYC‐Tip60 complex was purified using anti‐MYC magnetic beads, and the HAT activity of Tip60 was determined by incubating 50 g of MYC‐Tip60 complex using HAT assays. The data are presented as mean ± SD. (C) KYSE30 cells overexpressed FAM135B and related controls for 48 h and were then treated with or without 10‐μM BLM for 12 h. Cell lysates were subjected to immunoprecipitation (IP) analysis. (D) KYSE510 cells were transfected with siFAM135B and related controls for 48 h, then treated with or without 10‐μM BLM for 12 h. Cell lysates were subjected to IP analysis. Pan‐AC at an identical molecular weight as ATM indicated ATM acetylation. (E and F) KYSE30 cells with overexpressed FAM135B (E) and KYSE510 cells transfected with siFAM135B (F), and their related controls for 48 h were then treated with or without 10‐μM BLM for 12 h, then cells were harvested for western blotting. Pan‐AC at an identical molecular weight as ATM indicated ATM acetylation, and H4K9AC indicated TIP60 activity. (G) HEK‐293T cells transfected with FAM135B‐FLAG, TIP60 siRNA and their controls as indicated for 48 h were treated with 10‐μM BLM for 12 h, then cells were harvested for western blot analysis. (H) FAM135B's effects on the DNA damage response (DDR) are TIP60‐dependent, KYSE30 cells transfected with TIP60 siRNA, FLAG‐FAM135B and their controls as indicated for 48 h, then treated with 10‐μM BLM for 12 h, then cells were harvested for western blot analysis. (I) FAM135B's effects on the DDR are TIP60‐dependent, KYSE510 cells transfected with TIP60 siRNA, FAM135B siRNA and their controls for 48 h were treated with 10‐μM BLM for 12 h, then cells were harvested for western blot analysis. The GAPDH antibody was used to monitor equal loading in western blots. Data are represented as the mean ± SD from three independent experiments, *p < .05.