Figure 3.
Figure 3. HDAC6 knock-down increases CD20 levels and potentiates the efficacy of rituximab in vitro and in vivo. Raji cells were stably transduced with either pLKO.1 or pLKO.1-shHDAC6. (A) HDAC6 levels were assessed with qRT-PCR with SYBRGreen. The experiments were repeated independently twice. (B) The levels of HDAC6 and HDAC1 were assessed in western blotting. β-actin levels were used as a loading control. The experiments were repeated independently twice. (C) The levels of CD20 and acetylated-tubulin were assessed in western blotting. β-actin level was used as a loading control. Densitometric analysis from 4 independent immunoblots was performed using Image Studio Lite. The results are presented as a fold change of band intensities vs controls (± SEM). Statistical significance was determined with Mann-Whitney test, *P < .05 vs control. (D) 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 MFI of control cells (± SD). Statistical significance was determined with Mann-Whitney test, *P < .05 vs control. The experiments were repeated independently 3 times. (E) R-CDC and O-CDC were performed as described earlier (Figure 1D). Statistical significance was determined using 2-way analysis of variance test with Tukey’s correction, ****P < .0001 vs control. The experiments were repeated independently 3 times. (F-G) For in vivo studies, BALB/c SCID mice were inoculated subcutaneously with Raji cells either stably transduced with scrambled shRNA (pLKO.1) or shRNA against HDAC6 (pLKO.1-shHDAC6). Mice (n = 5-10) were then injected intraperitoneally with rituximab (30 mg/kg) 3 times a week for 3 consecutive weeks. (F) Effect of rituximab on tumor growth. Statistical significance was determined using multiple unpaired t tests, *P < .05, RTX pLKO.1 vs RTX shHDAC6. (G) Kaplan-Meier survival plot of mice pooled from 2 independent experiments. Statistical significance was determined using log-rank survival analysis, ****P < .0001, RTX pLKO.1 vs RTX shHDAC6.

HDAC6 knock-down increases CD20 levels and potentiates the efficacy of rituximab in vitro and in vivo. Raji cells were stably transduced with either pLKO.1 or pLKO.1-shHDAC6. (A) HDAC6 levels were assessed with qRT-PCR with SYBRGreen. The experiments were repeated independently twice. (B) The levels of HDAC6 and HDAC1 were assessed in western blotting. β-actin levels were used as a loading control. The experiments were repeated independently twice. (C) The levels of CD20 and acetylated-tubulin were assessed in western blotting. β-actin level was used as a loading control. Densitometric analysis from 4 independent immunoblots was performed using Image Studio Lite. The results are presented as a fold change of band intensities vs controls (± SEM). Statistical significance was determined with Mann-Whitney test, *P < .05 vs control. (D) 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 MFI of control cells (± SD). Statistical significance was determined with Mann-Whitney test, *P < .05 vs control. The experiments were repeated independently 3 times. (E) R-CDC and O-CDC were performed as described earlier (Figure 1D). Statistical significance was determined using 2-way analysis of variance test with Tukey’s correction, ****P < .0001 vs control. The experiments were repeated independently 3 times. (F-G) For in vivo studies, BALB/c SCID mice were inoculated subcutaneously with Raji cells either stably transduced with scrambled shRNA (pLKO.1) or shRNA against HDAC6 (pLKO.1-shHDAC6). Mice (n = 5-10) were then injected intraperitoneally with rituximab (30 mg/kg) 3 times a week for 3 consecutive weeks. (F) Effect of rituximab on tumor growth. Statistical significance was determined using multiple unpaired t tests, *P < .05, RTX pLKO.1 vs RTX shHDAC6. (G) Kaplan-Meier survival plot of mice pooled from 2 independent experiments. Statistical significance was determined using log-rank survival analysis, ****P < .0001, RTX pLKO.1 vs RTX shHDAC6.

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