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SIRT6 deacetylates PKM2 to suppress its nuclear localization and oncogenic functions - PubMed

  • ️Fri Jan 01 2016

SIRT6 deacetylates PKM2 to suppress its nuclear localization and oncogenic functions

Abhishek Bhardwaj et al. Proc Natl Acad Sci U S A. 2016.

Abstract

SIRT6 (sirtuin 6) is a member of sirtuin family of deacetylases involved in diverse processes including genome stability, metabolic homeostasis, and tumorigenesis. However, the role of SIRT6 deacetylase activity in its tumor-suppressor functions is not well understood. Here we report that SIRT6 binds to and deacetylates nuclear PKM2 (pyruvate kinase M2) at the lysine 433 residue. PKM2 is a glycolytic enzyme with nonmetabolic nuclear oncogenic functions. SIRT6-mediated deacetylation results in PKM2 nuclear export. We further have identified exportin 4 as the specific transporter mediating PKM2 nuclear export. As a result of SIRT6-mediated deacetylation, PKM2 nuclear protein kinase and transcriptional coactivator functions are abolished. Thus, SIRT6 suppresses PKM2 oncogenic functions, resulting in reduced cell proliferation, migration potential, and invasiveness. Furthermore, studies in mouse tumor models demonstrate that PKM2 deacetylation is integral to SIRT6-mediated tumor suppression and inhibition of metastasis. Additionally, reduced SIRT6 levels correlate with elevated nuclear acetylated PKM2 levels in increasing grades of hepatocellular carcinoma. These findings provide key insights into the pivotal role of deacetylase activity in SIRT6 tumor-suppressor functions.

Keywords: PKM2; SIRT6; deacetylation; tumor suppressor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

SIRT6 interacts with PKM2. (A) HepG2 cells were infected with adenovirus expressing GFP (Ad-GFP) or SIRT6 tagged with FLAG and HA epitopes (Ad-SIRT6). Cells were harvested, and nuclear extracts were sequentially immunoprecipitated with FLAG and HA antibody affinity resins. The SIRT6-associated proteins were detected by SDS/PAGE and silver staining. (B) HepG2 cells were transfected with the PKM2 construct. Six hours posttransfection, the cells were infected with Ad-GFP or Ad-SIRT6. Twenty-four hours postinfection cell were harvested and subjected to immunoprecipitation using anti-SIRT6 antibody (Left) or anti-PKM2 antibody (Right). Western blots then were performed for the indicated proteins. (C, Left) GST, GST-SIRT6, and His-PKM2 were bacterially expressed and purified. (Right) The GST pull-down assay was performed followed by Western blotting for the indicated proteins. (D) HepG2 cells were subjected to glucose starvation for the indicated periods. The cells then were harvested, and Western blotting was performed for the indicated proteins. (E) HepG2 cells were subjected to glucose starvation for the indicated periods. The cells then were harvested, and Western blotting was performed for the indicated proteins from nuclear and cytoplasmic fractions. (F) HepG2 cells were subjected to glucose starvation for the indicated periods. Nuclear extracts were subjected to immunoprecipitation using anti-SIRT6 antibody (Left) or anti-PKM2 antibody (Right), and Western blotting was performed for the indicated proteins. (G) HepG2 cells were transfected with constructs expressing full-length or different domains of PKM2 as the GST fusion protein. Six hours posttransfection, the cells were infected with Ad-GFP or Ad-SIRT6. Twenty-four hours postinfection, cell lysates were subjected to GST pull down followed by immunoblotting for the indicated proteins. (H) HepG2 cells were transfected with constructs expressing full-length or different domains of SIRT6 as the GST fusion protein. Six hours posttransfection, the cells were infected with adenovirus expressing GFP (Ad-GFP) or PKM2 tagged with FLAG epitope (Ad-PKM2). Twenty-four hours postinfection, cell lysates were subjected to GST pull down followed by immunoblotting for the indicated proteins.

Fig. 2.
Fig. 2.

SIRT6 deacetylates PKM2 at the Lys433 residue. (A) AcK433-PKM2 peptide was incubated either alone (control) or in presence of recombinant SIRT6, and the peptide molecular mass was determined by MS. The relative positions of acetylated (1,441.72 Da) and deacetylated (1,399.70 Da) PKM2 peptides are shown. (B) Results of SIRT6 deacetylation reactions using acetylated PKM2 peptides. The peptide molecular mass was determined by MS as in A. (C) HepG2 cells were stably transfected (pooled zeocin-resistant population) with control (scrambled) or SIRT6 shRNA. HepG2 control (HepG2) and SIRT6kd (HepG2 SIRT6kd) cells were subjected to glucose starvation for the indicated periods. Cells then were harvested, and Western blotting was performed for the indicated proteins. (D) HepG2 control (HepG2) and SIRT6kd (HepG2 SIRT6kd) cells were subjected to glucose starvation for the indicated periods. HepG2 SIRT6kd cells were infected with Ad-SIRT6 or with adenovirus expressing the deacetylase-dead SIRT6 mutant (Ad-SIRT6H133Y) during the last 24 h of the 36-h period as indicated. The cells then were harvested, and Western blotting was performed for the indicated proteins from nuclear and cytoplasmic fractions. (E) HepG2 control (HepG2) and SIRT6kd (HepG2 SIRT6kd) cells were subjected to glucose starvation for the indicated periods. HepG2 SIRT6kd cells were infected with Ad-SIRT6 or Ad-SIRT6H133Y during the last 24 h of the 36-h period as indicated. Immunofluorescence staining was performed for the indicated proteins. DAPI was used to counterstain the nucleus. (F) HepG2 cells were transfected with the FLAG-tagged PKM2K433Q construct and were subjected to glucose starvation for the indicated periods posttransfection. The cells then were harvested, and Western blotting was performed for the indicated proteins from nuclear and cytoplasmic fractions. (G) HepG2 cells were transfected with the FLAG-tagged PKM2K433R construct and were subjected to glucose starvation for the indicated periods posttransfection. The cells then were harvested, and Western blotting was performed for the indicated proteins from nuclear and cytoplasmic fractions.

Fig. S1.
Fig. S1.

SIRT6 modulates PKM2 protein kinase activity and coactivator functions upon serum starvation. (A) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to serum starvation for the indicated periods. Cells then were harvested, and Western blotting was performed for the indicated proteins. (B) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to serum starvation for the indicated periods. Total RNA was isolated, and relative mRNA levels were analyzed by RT-qPCR for the indicated genes. Error bars represent means ± SD of three independent experiments with triplicate samples. (C) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to serum starvation for the indicated periods. Total RNA was isolated, and relative mRNA levels were analyzed by RT-qPCR for the indicated genes. Error bars represent means ± SD of three independent experiments with triplicate samples.

Fig. S2.
Fig. S2.

SIRT6 suppresses PKM2 oncogenic functions in other cell types. (A) H1299 cells were stably transfected (pooled zeocin-resistant population) with control (scrambled) shRNA, SIRT6 shRNA, or SIRT6 shRNA along with PKM2 shRNA. H1299 control (H1299), SIRT6kd (H1299 SIRT6kd), and SIRT6/PKM2 double-knockdown (H1299 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. Cells then were harvested, and Western blotting was performed for the indicated proteins. (B) H1299 control (H1299), SIRT6kd (H1299 SIRT6kd), and SIRT6/PKM2 double-knockdown (H1299 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. Total RNA was isolated, and relative mRNA levels were analyzed by RT-qPCR for the indicated genes. Error bars represent means ± SD of three independent experiments with triplicate samples. (C) H1299 control (H1299), SIRT6kd (H1299 SIRT6kd), and SIRT6/PKM2 double-knockdown (H1299 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. Total RNA was isolated, and relative mRNA levels were analyzed by RT-qPCR for the indicated genes. Error bars represent means ± SD of three independent experiments with triplicate samples. (D) H1299 SIRT6kd cells (gray bars) or SIRT6/PKM2 double-knockdown cells (blue bars) were stably transfected (pooled hygromycin-resistant population) with a dual expression plasmid encoding FLAG-tagged wild-type SIRT6, FLAG-tagged SIRT6H133Y, PKM2K433Q, and FLAG-tagged wild-type SIRT6 along with PKM2K433Q or PKM2K433R as indicated. H1299 SIRT6kd or SIRT6/PKM2 double-knockdown cells stably transfected with empty vector served as control. The cells were harvested and counted at the indicated time points. Error bars represent means ± SD of three independent experiments with duplicate samples. (E) The migration potential of the cells in D was measured as the percentage of cells migrating to the lower chamber. Error bars represent means ± SD of three independent experiments with triplicate samples. (F) The in vitro invasion of the cells in D was measured as the percentage of cells migrating to the lower chamber. Error bars represent means ± SD of three independent experiments with duplicate samples. (G) Primary MEFs were transduced with control (scrambled) shRNA, SIRT6 shRNA, or SIRT6 shRNA along with PKM2 shRNA. MEF control (MEF), SIRT6kd (MEF SIRT6kd), and SIRT6/PKM2 double-knockdown (MEF SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. Cells then were harvested, and Western blotting was performed for the indicated proteins. (H) MEF control (MEF), SIRT6kd (MEF SIRT6kd), and SIRT6/PKM2 double-knockdown (MEF SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. Total RNA was isolated, and relative mRNA levels for the indicated genes were analyzed by RT-qPCR. Error bars represent means ± SD of three independent experiments with triplicate samples. (I) MEF control (MEF), SIRT6kd (MEF SIRT6kd), and SIRT6/PKM2 double-knockdown (MEF SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. Total RNA was isolated, and relative mRNA levels were analyzed by RT-qPCR for the indicated genes. Error bars represent means ± SD of three independent experiments with triplicate samples.

Fig. S3.
Fig. S3.

The deacetylase-dead SIRT6 mutant (SIRT6H133Y) cannot repress PKM2 functions. (A) HepG2 SIRT6kd (HepG2 SIRT6kd) cells were subjected to glucose starvation for 36 h and were infected with adenovirus expressing SIRT6 (Ad-SIRT6) or adenovirus expressing the deacetylase-dead SIRT6 mutant (Ad-SIRT6H133Y) during the last 24 h of the 36-h period, as indicated. The cells then were harvested, and Western blotting was performed for the indicated proteins. (B) HepG2 SIRT6kd (HepG2 SIRT6kd) cells were subjected to glucose starvation for 36 h and were infected with Ad-SIRT6 or Ad-SIRT6H133Y during the last 24 h of the 36-h period, as indicated. The cells then were harvested, and Western blotting was performed for the indicated proteins. (C) HepG2 SIRT6kd (HepG2 SIRT6kd) cells were subjected to glucose starvation for 36 h and were infected with Ad-SIRT6 or Ad-SIRT6H133Y during the last 24 h of the 36-h period as indicated. The cells then were harvested, and nuclear extracts were subjected to immunoprecipitation using anti-PKM2 antibody. Western blotting was performed for the indicated proteins.

Fig. 3.
Fig. 3.

Deacetylated nuclear PKM2 interacts with XPO4, leading to its nuclear export. (A) HepG2 cells were stably transfected (pooled zeocin-resistant population) with shRNA targeting XPO1, XPO4, XPO7, or their respective control (scrambled) shRNA. HepG2 XPO1kd (Left), XPO4kd (HepG2 XPO4kd) (Center), XPO7kd (HepG2 XPO7kd) (Right), and their respective control (HepG2) cells were subjected to glucose starvation for the indicated periods. Cells then were harvested, and Western blotting was performed for the indicated proteins from nuclear extracts. (B) HepG2 cells were subjected to glucose starvation for 24 h. Nuclear extracts were subjected to immunoprecipitation using anti-XPO4 antibody (Left) or anti-PKM2 antibody (Right), and Western blotting was performed for the indicated proteins. (C) HepG2 PKM2kd cells were subjected to glucose starvation for 24 h and were infected with control adenovirus (Ad-GFP), adenovirus expressing PKM2 (Ad-PKM2), or adenovirus expressing the PKM2K433Q mutant (Ad-PKM2K433Q) during the last 12 h of the 24-h period as indicated. Nuclear extracts were subjected to immunoprecipitation using anti-XPO4 antibody (Left) or anti-PKM2 antibody (Right), and Western blotting was performed for the indicated proteins. (D) HepG2 SIRT6kd cells were subjected to glucose starvation for 24 h and were infected with control adenovirus (Ad-GFP), adenovirus expressing FLAG-tagged SIRT6 (Ad-SIRT6), or adenovirus expressing FLAG-tagged SIRT6H133Y mutant (Ad-SIRT6H133Y) during the last 12 h of the 24-h period as indicated. Nuclear extracts were subjected to immunoprecipitation using anti-XPO4 antibody (Left) or anti-PKM2 antibody (Right), and Western blotting was performed for the indicated proteins.

Fig. 4.
Fig. 4.

SIRT6 modulates PKM2 protein kinase activity. (A) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. Cells then were harvested, and Western blotting was performed for the indicated proteins. (B) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. Relative mRNA levels were analyzed by quantitative RT-PCR (RT-qPCR) for the indicated genes. Error bars represent means ± SD of three independent experiments with triplicate samples. (C) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. A ChIP assay then was performed with control IgG or STAT3 antibody. Error bars represent means ± SD of three independent experiments with triplicate samples. (D) Part of the chromatin immunoprecipitated with STAT3 antibody in 4C was again subjected to ChIP using control IgG or PKM2 antibody. Error bars represent means ± SD of three independent experiments with triplicate samples.

Fig. S4.
Fig. S4.

SIRT6 occupancy at STAT3 and SIRT6 target promoters. (A) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. A ChIP assay then was performed with control IgG or SIRT6 antibody. The fold enrichment of coprecipitating DNA was determined by qPCR for the indicated promoters. Error bars represent means ± SD of three independent experiments with triplicate samples. (B) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), PKM2kd (HepG2 PKM2kd), and PKM2kd along with PKM2K433Q-expressing [HepG2 PKM2kd (+PKM2K433Q)] cells were subjected to glucose starvation for the indicated periods. A ChIP assay then was performed with control IgG or SIRT6 antibody. The fold enrichment of coprecipitating DNA was determined by qPCR for the indicated promoters. Error bars represent means ± SD of three independent experiments with triplicate samples. (C) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), PKM2kd (HepG2 PKM2kd), and PKM2kd along with PKM2K433Q-expressing [HepG2 PKM2kd (+PKM2K433Q)] cells were subjected to glucose starvation for the indicated periods. A ChIP assay then was performed with control IgG or AcK56-H3 antibody. The fold enrichment of coprecipitating DNA was determined by qPCR for the indicated promoters. Error bars represent means ± SD of three independent experiments with triplicate samples. (D) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), PKM2kd (HepG2 PKM2kd), and PKM2kd along with PKM2K433Q-expressing [HepG2 PKM2kd (+PKM2K433Q)] cells were subjected to glucose starvation for the indicated periods. A ChIP assay then was performed with control IgG or AcK9-H3 antibody. The fold enrichment of coprecipitating DNA was determined by qPCR for the indicated promoters. Error bars represent means ± SD of three independent experiments with triplicate samples. (E) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), PKM2kd (HepG2 PKM2kd), and PKM2kd along with PKM2K433Q-expressing [HepG2 PKM2kd (+PKM2K433Q)] cells were subjected to glucose starvation for the indicated periods. Total RNA was isolated, and relative mRNA levels were analyzed by RT-qPCR for the indicated genes. Error bars represent means ± SD of three independent experiments with triplicate samples.

Fig. 5.
Fig. 5.

SIRT6 abrogates PKM2–β-catenin interaction. (A and B) HepG2 control (HepG2) and SIRT6kd (HepG2 SIRT6kd) cells were subjected to glucose starvation for the indicated periods. Nuclear extracts were subjected to immunoprecipitation using anti-PKM2 antibody (A) or anti–β-catenin antibody (B), and Western blotting was performed for the indicated proteins. (C) HepG2 control (HepG2), SIRT6kd knockdown (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. Relative mRNA levels were analyzed by RT-qPCR for the indicated genes. Error bars represent means ± SD of three independent experiments with triplicate samples. (D) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. A ChIP assay then was performed with control IgG or β-catenin antibody. Error bars represent means ± SD of three independent experiments with triplicate samples. (E) Part of the chromatin immunoprecipitated with β-catenin antibody in D was again subjected to ChIP using control IgG or PKM2 antibody. Error bars are means ± SD of three independent experiments with triplicate samples.

Fig. S5.
Fig. S5.

SIRT6 does not affect H3K9ac and H3K56ac levels at β-catenin target promoters. (A) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. A ChIP assay then was performed with control IgG or SIRT6 antibody. The fold enrichment of coprecipitating DNA was determined by qPCR for the indicated promoters. Error bars represent means ± SD of three independent experiments with triplicate samples. (B) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. A ChIP assay then was performed with control IgG or AcK56-H3 antibody. The fold enrichment of coprecipitating DNA was determined by qPCR for the indicated promoters. Error bars represent means ± SD of three independent experiments with triplicate samples. (C) HepG2 control (HepG2), SIRT6kd (HepG2 SIRT6kd), and SIRT6/PKM2 double-knockdown (HepG2 SIRT6kd/PKM2kd) cells were subjected to glucose starvation for the indicated periods. A ChIP assay then was performed with control IgG or AcK9-H3 antibody. The fold enrichment of coprecipitating DNA was determined by qPCR for the indicated promoters. Error bars represent means ± SD of three independent experiments with triplicate samples.

Fig. 6.
Fig. 6.

SIRT6 suppresses PKM2-dependent cell proliferation and malignant phenotype. (A) HepG2 SIRT6kd cells (gray bars) or SIRT6/PKM2 double-knockdown cells (blue bars) were stably transfected (pooled hygromycin-resistant population) with a dual expression plasmid encoding FLAG-tagged wild-type SIRT6, FLAG-tagged SIRT6H133Y, PKM2K433Q, and FLAG-tagged wild-type SIRT6 along with PKM2K433Q or PKM2K433R as indicated. HepG2 SIRT6kd or SIRT6/PKM2 double-knockdown cells stably transfected with empty vector served as control. The cells were harvested and counted at the indicated time points. Error bars represent means ± SD of three independent experiments with duplicate samples. (B) The glucose uptake of the cells in A was measured. Error bars represent means ± SD of three independent experiments with duplicate samples. (C) The lactate production of the cells in A was measured. Error bars represent means ± SD of three independent experiments with duplicate samples. (D) The migration potential of the cells in A was measured as the percentage of cells migrating to the bottom chamber. Error bars represent means ± SD of three independent experiments with triplicate samples. (E) In vitro invasion of the cells in A was measured as the percentage of cells migrating to the bottom chamber. Error bars represent means ± SD of three independent experiments with duplicate samples.

Fig. S6.
Fig. S6.

Expression levels of various SIRT6 and PKM2 constructs in stable cell lines and in xenograft tumors and quantitative analysis of SIRT6 and AcK433-PKM2 levels in human liver carcinoma. (A) HepG2 SIRT6kd or SIRT6/PKM2 double-knockdown cells (a pooled hygromycin-resistant population) were stably transfected with a dual expression plasmid encoding FLAG-tagged wild-type SIRT6, FLAG-tagged SIRT6H133Y, PKM2K433Q, FLAG-tagged wild-type SIRT6 along with wild-type PKM2 or PKM2K433Q or PKM2K433R as indicated. HepG2 SIRT6 knockdown or SIRT6/PKM2 double-knockdown cells stably transfected with empty vector served as control. The cells were harvested, and Western blotting was performed for the indicated proteins. (B) Tumor lysates (Fig. 7B) were prepared and analyzed by immunoblotting for the indicated proteins. The data shown are representative of three independent experiments. (C) Quantitation of SIRT6 levels in different grades of human liver carcinoma normalized with respect to matched normal adjacent tissue. The average signal intensity (in arbitrary units, AU) from four random fields was used for the analysis. The data shown are representative of three independent experiments. Error bars represent means ± SD. (D) Quantitation of AcK433-PKM2 levels in different grades of human liver carcinoma normalized with respect to matched normal adjacent tissue. The average signal intensity (in arbitrary units, AU) from four random fields was used for the analysis. The data shown are representative of three independent experiments. Error bars represent means ± SD. (E) Analysis of the correlation between SIRT6 (in C) and AcK433-PKM2 (in D) levels in human liver carcinoma (n = 48; Spearman's coefficient r = −0.8882, P < 0.0001).

Fig. 7.
Fig. 7.

PKM2 deacetylation is integral to SIRT6 tumor-suppressor functions. (A and B) HepG2 SIRT6kd cells (dashed lines) or SIRT6/PKM2 double-knockdown cells (solid lines) were stably transfected (pooled hygromycin-resistant population) with a dual expression plasmid encoding FLAG-tagged wild-type SIRT6, FLAG-tagged SIRT6H133Y, PKM2K433Q, and FLAG-tagged wild-type SIRT6 along with PKM2K433Q or PKM2K433R as indicated. These cells were s.c. injected into the right flank of nude mice. HepG2 SIRT6kd or SIRT6/PKM2 double-knockdown cells stably transfected with empty vector were used as control. (A) Tumor volume was measured on the indicated days. The data shown are representative of three independent experiments (n = 5 mice per group). Error bars represent means ± SD from five individual mice. (B) At the end of 30 d, tumors were excised and weighed. The data shown are representative of three independent experiments (n = 5 mice per group). Error bars represent means ± SD from five individual mice. (C) HepG2Luc2 cells were stably transfected (pooled hygromycin-resistant population) with a dual expression plasmid encoding wild-type SIRT6 and wild-type SIRT6 along with PKM2K433Q. HepG2Luc2 cells stably transfected with empty vector were used as control. These cells were injected into the liver of nude mice. Bioluminescence imaging was performed weekly; representative images are shown. The data shown are representative of three independent experiments using five individual mice per group. (D) Bioluminescence quantification of the cells in C was performed at the indicated time points. The data shown are representative of three independent experiments (n = 5 mice per group). Error bars represent means ± SD from five individual mice. (E) At the end of 4 wk, lungs and pancreas were collected from the mice orthotopically implanted with cells expressing wild-type SIRT6 along with PKM2K433Q as shown in C, and ex vivo imaging was performed to examine spontaneous metastases in lungs (Left) and pancreas (Right). The data shown are representative of three independent experiments using five individual mice per group. (F) At the end of 4 wk, lysates of primary orthotopic tumors obtained in C were analyzed by immunoblotting for the indicated proteins. The data shown are representative of three independent experiments. (G) Representative images of immunostaining of SIRT6 and AcK433-PKM2 in different grades of human liver carcinoma (HCC) and matched normal adjacent tissue (NAT) sections. DAPI was used to counter stain nucleus.

Fig. S7.
Fig. S7.

Protein kinase activity of PKM2 purified from nuclear and cytoplasmic fractions. Phosphorylation of purified STAT3 by PKM2 immunopurified from nuclear (NE) and cytoplasmic (CE) fractions of HepG2 cells was analyzed by Western blotting for the indicted proteins.

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