Specific HDAC6 inhibitors upregulate CD20 levels and potentiate the efficacy of anti-CD20 mAbs. (A-C) Raji cells were preincubated for 48 hours with increasing concentrations of HDAC6-specific inhibitors: tubacin, tubastatin A, and ricolinostat. (A) The levels of surface CD20 were analyzed with flow cytometry on staining with FITC-conjugated anti-CD20 antibody. Cell viability was assessed with PI staining. The results are presented as a percentage of mean fluorescence intensity (MFI) of control cells (± standard deviation [SD]). Statistical significance was determined with Kruskal-Wallis test, **P < .01 vs controls. The experiments were repeated independently 3 times. (B) The levels of CD20 and acetylated-tubulin (a marker of HDAC6 inhibition) were assessed with western blotting in whole-cell lysates. β-actin was used as loading control. Densitometric analysis from 5 independent immunoblots was performed using Image Studio Lite. The results are presented as a fold change of band intensities vs controls (± standard error of the mean [SEM]). Statistical significance was determined with Mann-Whitney test, *P < .05 vs controls. (C) Equal amounts of control and drug-pretreated cells were incubated for 60 minutes with rituximab (R-CDC) or ofatumumab (O-CDC; 1-100 µg/mL) and 10% human AB serum as a source of complement. Cell viability was assessed with PI staining. The survival of cells is presented as a percentage of control cells without antibody (± SD). Statistical significance was determined using 2-way analysis of variance test with Tukey’s correction, **P < .01, ***P < .001, ****P < .0001 vs controls. The experiments were repeated independently 3 times.