SIRT2-mediated inactivation of p73 is required for glioblastoma tumorigenicity (2018)
Figure 3

Figure 3. p73 is required for SIRT2 knockdown-induced suppression of glioblastoma proliferation.

A Schematic representation of p73 isoforms. TAD, transactivation domain; DBD, DNA-binding domain; OD, oligomerization domain; SAM, sterile alpha motif domain. B Immunoblotting analysis of p73 in GB2 cells infected with a control (Empty) or shTP73-expressing lentivirus for 96 h.

C Growth curve of GB16 cells infected with the indicated lentiviruses. Bars indicate mean ± s.d. (n = 3).

D Ectopic expression of the ∆N isoform of p73β suppresses the expression levels of PUMA and GADD45 induced by SIRT2 knockdown in GB2 cells. Four days after lentiviral infection, the expression levels of PUMA and GADD45 mRNA were measured by qRT-PCR. Bars indicate mean ± s.d. (n = 3).

E GB2 and GB4 cells [5 × 104 and 2 × 104 cells, respectively (indicated by the dashed lines)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± s.d. (n = 4).

F HEK293 cells were transfected with TAp73α along with ΔNp73β. PUMA mRNA was measured by qRT-PCR analyses. Bars indicate mean ± s.d. (n = 3).

G GB2 cells (5 × 104 cells, dashed line) were infected with the indicated lentiviruses, respectively. After 96 h, the number of viable cells was counted. shTP73 corresponds to both α and β isoforms of p73. Bars indicate mean ± s.d. (n = 4).

H GB2 cells were transfected with TAp73α along with NLS-SIRT2 (nuclear-localizing mutant of SIRT2) or 3mut (deacetylase-inactive, nuclear-localizing mutant of SIRT2) and a reporter construct consisting of the promoter region of PUMA fused to a luciferase gene (Left panel). Reporter activities were determined by Dual luciferase assays (Right panel). Bars indicate mean ± s.d. (n = 3).

I GB2 cells were electroporated with TAp73α along with NLS-SIRT2 (nuclear-localizing mutant of SIRT2) or 3mut (deacetylase-inactive, nuclear-localizing mutant of SIRT2). The expression of PUMA and GADD45 was measured by qRT-PCR analysis. Bars indicate mean ± s.d. (n = 3)

Data information: Statistical significance were evaluated using unpaired two-tailed t-test. *p < 0.05; **P < 0.01.

Figure. 4. SIRT2 deacetylates p73 and suppresses its transcriptional activity.

A Cells transfected with FLAG-tagged p73 isoforms were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.

B Three conserved lysine residues are located in the most C-terminal region of p73. Amino acid sequences of p73 of the indicated species and human p53 and p63 are aligned. *, conserved and acetylated lysine residues, black frame, highly conserved residues, gray frame, conserved residues.

C Schematic representation of TAp73α and K3R. K3R is a mutant TAp73α in which three lysine (K) residues in the C-terminal region are replaced with arginine (R).

D Cells transfected with the indicated constructs were treated with 20 μM AGK2 or DMSO, respectively, for 6 h and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting with antibody against acetylated lysine or FLAG.

E HEK293T cells transfected with FLAG-tagged TAp73α were treated with 20 μM AGK2 (pre-AGK2 +) or DMSO (pre-AGK2 -) for 6 h. p73 was purified by immunoprecipitation and incubated with recombinant SIRT2 (10 U), NAD (1 mM) and/or AGK2 (3.5 μM) as indicated.

F qRT-PCR analysis of PUMA and GADD45 mRNA in GB2 cells infected with the indicated lentiviruses. Bars indicate mean ± s.e.m. (n = 4-5).

G Expression of p73 in (F) was determined by immunoblotting with anti-p73 antibody. RFP was used as a control.

H GB glioblastima neurospheres [5 × 104 cells (indicated by the dashed line)] were infected with the indicated lentiviruses. After 96 h, the number of viable cells was counted. Bars indicate mean ± s.d. (n = 4).

I GB2 cells infected with the indicated lentiviruses were intracranially transplanted into immunocompromised mice. After 8 weeks, mice (3 to 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT-PCR.

J SIRT2-mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells. SIRT2 regulates the transcriptional activity of the tumor suppressor p73 by deacetylating its C-terminal lysine residues.

Data information: Statistical significance were evaluated using unpaired two-tailed t-test. *p < 0.05; **P < 0.01.

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