American Society of Hematology

Figure 6.

$HDAC6 inhibition increases CD20 synthesis de novo. (A) Raji cells were coincubated for 24 hours with 10 µM tubacin (TUB) and inhibitors of protein translation: 10 µg/mL cycloheximide (CHX) or 200 nM homoharringtonine (HOMO). The levels of surface CD20 were analyzed with flow cytometry as described in Figure 1A. The results are presented as a percentage of MFI of control cells (± SD). Statistical significance was determined with Kruskal-Wallis test, *P < .05 vs controls. The experiments were repeated independently 3 times. (B) Raji cells were coincubated with TUB, CHX, or HOMO as described in Figure 6A. CD20 levels in whole-cell lysates were assessed with western blotting. β-actin was used as 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 controls. (C-D) Raji cells were coincubated for 4 hours with 50 µM Click-IT AHA and 10 µM tubacin and inhibitors of protein translation cycloheximide (100 µg/mL) or homoharringtonine (2 µM). (C) Newly synthesized proteins were labeled with biotin and immunoprecipitated using Neutravidin Aggarose beads. CD20 levels were assessed in biotinylated fraction, using western blotting. β-actin in input samples served as a loading control. Densitometric analysis from 3 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 controls. (D) Control, CHX, or tubacin-treated cells were analyzed with flow cytometry on staining of newly synthesized proteins with Alexa Fluor 488. The results are presented as a percentage of MFI of control cells (± SD). Statistical significance was determined with Kruskal-Wallis test, *P < .05 vs controls. The experiments were repeated independently twice. (E) Raji cells were coincubated for 4 hours with 50 µM Click-IT AHA and 10 µM tubacin. Newly synthesized proteins were labeled with biotin. Global de novo protein synthesis in control and tubacin-treated cells was assessed by western blotting with anti-biotin antibody. The experiments were repeated independently twice. (F-G) Control and tubacin-pretreated Raji cells were lysed in 25 mM KCl buffer, and the cytoplasmic fraction was separated onto a 10%-40% sucrose gradient. (F) Polysomal profiling of control and tubacin-treated cells. (G) Cytoplasmic RNA and polysomal-associated RNA from control or tubacin-pretreated Raji cells were reverse transcribed. CD20 levels were quantified by real-time polymerase chain reaction, using SYBR Green, and normalized to HPRT1 mRNA. qPCR analysis was performed in triplicates. Analysis of mRNA levels for each target was normalized to HPRT1 mRNA. The cytoplasmic RNA (totRNA) and the polysomal-associated RNA (polyRNA) ratios were determined as described in “Materials and methods.” The results are representative of the average RNA ratio ± SEM from 3 independent experiments. Statistical significance was determined with Kruskal-Wallis test, *P < .05 compared with total RNA.$

HDAC6 inhibition increases CD20 synthesis de novo. (A) Raji cells were coincubated for 24 hours with 10 µM tubacin (TUB) and inhibitors of protein translation: 10 µg/mL cycloheximide (CHX) or 200 nM homoharringtonine (HOMO). The levels of surface CD20 were analyzed with flow cytometry as described in Figure 1A. The results are presented as a percentage of MFI of control cells (± SD). Statistical significance was determined with Kruskal-Wallis test, *P < .05 vs controls. The experiments were repeated independently 3 times. (B) Raji cells were coincubated with TUB, CHX, or HOMO as described in Figure 6A. CD20 levels in whole-cell lysates were assessed with western blotting. β-actin was used as 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 controls. (C-D) Raji cells were coincubated for 4 hours with 50 µM Click-IT AHA and 10 µM tubacin and inhibitors of protein translation cycloheximide (100 µg/mL) or homoharringtonine (2 µM). (C) Newly synthesized proteins were labeled with biotin and immunoprecipitated using Neutravidin Aggarose beads. CD20 levels were assessed in biotinylated fraction, using western blotting. β-actin in input samples served as a loading control. Densitometric analysis from 3 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 controls. (D) Control, CHX, or tubacin-treated cells were analyzed with flow cytometry on staining of newly synthesized proteins with Alexa Fluor 488. The results are presented as a percentage of MFI of control cells (± SD). Statistical significance was determined with Kruskal-Wallis test, *P < .05 vs controls. The experiments were repeated independently twice. (E) Raji cells were coincubated for 4 hours with 50 µM Click-IT AHA and 10 µM tubacin. Newly synthesized proteins were labeled with biotin. Global de novo protein synthesis in control and tubacin-treated cells was assessed by western blotting with anti-biotin antibody. The experiments were repeated independently twice. (F-G) Control and tubacin-pretreated Raji cells were lysed in 25 mM KCl buffer, and the cytoplasmic fraction was separated onto a 10%-40% sucrose gradient. (F) Polysomal profiling of control and tubacin-treated cells. (G) Cytoplasmic RNA and polysomal-associated RNA from control or tubacin-pretreated Raji cells were reverse transcribed. CD20 levels were quantified by real-time polymerase chain reaction, using SYBR Green, and normalized to HPRT1 mRNA. qPCR analysis was performed in triplicates. Analysis of mRNA levels for each target was normalized to HPRT1 mRNA. The cytoplasmic RNA (totRNA) and the polysomal-associated RNA (polyRNA) ratios were determined as described in “Materials and methods.” The results are representative of the average RNA ratio ± SEM from 3 independent experiments. Statistical significance was determined with Kruskal-Wallis test, *P < .05 compared with total RNA.

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