Methylated ACTB regulates translation and cell adhesion. (A) Fibronectin adhesion assay was carried out on HCT-116 Ct, SETD3 KO#1, and SETD3 KO#2. Top: Representative images of adherent cells stained with Crystal violet. Bottom: Crystal violet-stained cells were dissolved in 2% SDS, and the absorbance at 550 nm was measured. Error bars are S.D. Statistical analysis was performed on three experimental repeats. (**) P < 0.01. (B) Same as in A in HCT116 SETD3 KO cells rescued with increasing amounts of FLAG-SETD3 WT. (C) Fibronectin adhesion assay was carried out in HCT116 cells stably expressing empty, FLAG-WT ACTB, or FLAG-Mut ACTB H73A, as indicated. Top: Representative images of adherent cells stained with Crystal violet. Bottom: Crystal violet-stained cells were dissolved in 2% SDS, and the absorbance at 550 nm was measured. Error bars are S.D. Statistical analysis was performed on three experimental repeats. (**) P < 0.01. (D) Translation assay performed on HCT-116 Ct and two SETD3 KOs cells (KO1 and KO2). Cells were incubated with puromycin, and then cell extracts were submitted to western blot to detect protein synthesis using anti-puromycin antibody. (E) Same as in D in HCT-116 Ct and SETD3 KO rescued with empty, FLAG-WT, and Y313A SETD3. (F) Same as in D in ACTB KO cells, stably expressing empty, FLAG-WT ACTB, or FLAG-ACTB H73A.