Fig 2: Model of PKCd-dependent apoptosis.Left, when PKCd is overexpressed, SIRT6 expression decreases, allowing KDM2A to demethylate H3K36, resulting in more condensed chromatin and inhibition of NHEJ and HR repair. This results in increased genomic instability and apoptosis. Right, depletion of PKCd increases the expression of SIRT6, which in turn leads to the displacement of KDM2A from chromatin. Accumulation of H3K36me2 results in a more open chromatin structure, increases NHEJ and HR repair, and promotes cell survival.

Fig 3: SIRT6 regulates KDM2A ribosylation in PKCd-depleted cell.(A) Top: Whole cell lysates of ParC5 shNT, shd110, and shd680 cells were immunoblotted with the indicated antibodies. Bottom: Densitometry of SIRT6 expression normalized to ß-actin. (B) Relative expression of SIRT6 mRNA in ParC5 shNT, shd110, and shd680 cells was assayed by qRT-PCR. (C) Top: ParC5 cells transfected with pEV or pPKCdFLAG were analyzed for SIRT6 expression by immunoblot analysis. Bottom: Densitometry of SIRT6 expression normalized to ß-actin. (D) Relative expression of SIRT6 mRNA in ParC5 overexpressing either pEV or pPKCdFLAG cells was assayed by qRT-PCR. Shown is data from at least three independent biological replicates. Data is shown as mean ± SEM. (E) Top: ParC5 shNT, shd110, and shd680 cells were fractionated into cytoplasm, nuclear and chromatin fractions, and each fraction was immunoblotted with the indicated antibodies. Bottom: Percentage of KDM2A in nuclear and chromatin were calculated. (F) Top: Immunoprecipitation of KDM2A from ParC5 shNT, shd110, and shd680 nuclear fractions using KDM2A antibody or normal IgG control, and immunoblotted using anti-mono-ADP-ribose (MAR) and KDM2A antibodies. Bottom: Densitometry of MAR-KDM2A normalized to KDM2A. Statistics represent one-way ANOVA followed by Dunnett’s multiple comparisons for (A), (B) and (F), unpaired t-test for (C) and (D), and two-way ANOVA followed by Dunnett’s multiple comparisons for (E). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.