Journal: Acta Pharmaceutica Sinica. B

Article Title: Dihydrotanshinone I preconditions myocardium against ischemic injury via PKM2 glutathionylation sensitive to ROS

doi: 10.1016/j.apsb.2022.07.006

Figure Lengend Snippet: DT induces the nuclear translocation of PKM2. (A) KEGG enrichment of results from an iTRAQ study. Proteins with a P value < 0.05 (unpaired two-tailed Student's t -test) were assigned as differential protein. KEGG enrichment was performed and the twenty-five most significantly regulated pathways are listed; (B) A volcano plot shows the mean difference of the protein plotted against the P value (unpaired two-tailed Student's t -test, P < 0.05). (C) mRNA level of Pkm2 was determined by quantitative PCR after treatment with DT for 4 h ( n = 6). (D) Immunoblot analysis of PKM2 in HL-1 total cell extracts after treatment with DT for 4 h ( n = 3). (E) Immunoblot analysis of PKM2 in HL-1 nuclear and cytoplasmic extracts after treatment with DT for 4 h ( n = 3). (F) PKM2 oligomerization in HL-1 cells after incubation with DT for 4 h ( n = 3). (G) PKM2 activity in HL-1 cells after treatment with DT for 4 h ( n = 5). (H) HL-1 cells were pre-treated with DT for 4 h, then, after washing, cells were incubated in glucose-free DMEM under 1% O 2 (OGD) for another 6 h and cell viability was measured by a CCK-8 assay ( n = 6). The results are expressed as the mean ± SEM, except the proteome study (an unpaired two-tailed Student's t -test was used). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, statistically not significant.

Article Snippet: Anti-PKM2 antibody (cat#4053), anti-Lamin A/C antibody (cat# 2032) and anti-HIF-1 α antibody (cat#36169) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Translocation Assay, Multiplex sample analysis, Two Tailed Test, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Activity Assay, CCK-8 Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dihydrotanshinone I preconditions myocardium against ischemic injury via PKM2 glutathionylation sensitive to ROS

doi: 10.1016/j.apsb.2022.07.006

Figure Lengend Snippet: DT stabilizes HIF-1 α . (A) IP analysis of the interaction between PKM2 and HIF-1 α after incubation for 4 h ( n = 3). (B) HIF-1 α protein in Pkm2 KO and Gfp KO cells treated with DT for 4 h ( n = 3). (C) IP analysis of the interaction between PKM2 and HIF-1 α in the nuclear after incubation for 4 h ( n = 3). (D) IP analysis of the ubiquitination level of HIF-1 α ( n = 3). (E) Immunoblot analysis of Flag–HIF–1 α in HEK293T cells (cycloheximide, CHX, 10 μg/mL) ( n = 3). (F) mRNA level of Hif1a was determined by quantitative PCR after treatment with DT (1 and 2 μmol/L) or vehicle for 4 h ( n = 6). (G) Hif1a luciferase activity in Pkm2 or Gfp KO stable cell lines treated with DT for 4 h ( n = 3). (H) mRNA level of Il10 , Pdk1 , Hmox1 and Vegfa in HL-1 cells with DT for 4 h ( n = 4). The results are expressed as the mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, statistically not significant.

Article Snippet: Anti-PKM2 antibody (cat#4053), anti-Lamin A/C antibody (cat# 2032) and anti-HIF-1 α antibody (cat#36169) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Incubation, Ubiquitin Proteomics, Western Blot, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Stable Transfection

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dihydrotanshinone I preconditions myocardium against ischemic injury via PKM2 glutathionylation sensitive to ROS

doi: 10.1016/j.apsb.2022.07.006

Figure Lengend Snippet: The preconditioning effect of DT depended on transient ROS generation. (A) Immunoblot analysis of nuclear PKM2 and HIF-1 α in HL-1 cells treated with DT or Mito-Tempol (Mito-T) for 4 h ( n = 3). (B) Endogenous HIF-1 α and PKM2 in H9C2 cells treated with DT or Mito-T for 4 h (Scale bar: 10 μm, n = 3). (C) IP analysis of the interaction between PKM2 and HIF-1 α in the nucleus ( n = 3). (D) Immunoblot analysis of HIF-1 α in HL-1 cells after treatment with H 2 O 2 for 4 h ( n = 3). (E) PKM2 oligomerization in HL-1 cells treated with H 2 O 2 for 4 h ( n = 3). (F) Endogenous HIF-1 α and PKM2 in H9C2 cells treated with H 2 O 2 for 4 h (Scale bar: 10 μm, n = 3). (G) IP analysis of the interaction between PKM2 and HIF-1 α in the nucleus after incubation for 4 h ( n = 3). (H) PKM2 activity in HL-1 cells treated with H 2 O 2 for 4 h ( n = 5). (I) Hif1a luciferase reporter in Pkm2 or Gfp KO stable cells treated with H 2 O 2 for 4 h ( n = 4). (J) mRNA level of Il10 , Pdk1 , Hmox1 and Vegfa in HL-1 cells treated with H 2 O 2 for 4 h ( n = 4). The results are expressed as the mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, statistically not significant.

Article Snippet: Anti-PKM2 antibody (cat#4053), anti-Lamin A/C antibody (cat# 2032) and anti-HIF-1 α antibody (cat#36169) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Incubation, Activity Assay, Luciferase

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dihydrotanshinone I preconditions myocardium against ischemic injury via PKM2 glutathionylation sensitive to ROS

doi: 10.1016/j.apsb.2022.07.006

Figure Lengend Snippet: (A) The GSSG/GSH ratio in HL-cells treated with DT for 4 h ( n = 4). (B) Immunoblot analysis of α -GSH after treatment with GSSG (1 and 2 mmol/L) for 4 h ( n = 3). (C) IP analysis of the binding of α -GSH to PKM2 ( n = 3). (D) Immunoblot analysis of HIF-1 α and PKM2 in the nuclear fractions ( n = 3). (E) HL-1 cells were pre-treated with GSSG, then an SA-GSSG pull down assay was performed and the pellet was detected by immunoblot ( n = 3). (F) The pull-down assay was performed in a recombinant PKM2 system ( n = 3). (G) Immunoblot analysis of HIF-1 α , PKM2 and GLRX1 in the nuclear fractions ( n = 3). (H) IP analysis of nuclear α -GSH and PKM2 in HL-1 cells treated with DT for 4 h ( n = 3). The results are expressed as the mean ± SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Anti-PKM2 antibody (cat#4053), anti-Lamin A/C antibody (cat# 2032) and anti-HIF-1 α antibody (cat#36169) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Binding Assay, Pull Down Assay, Recombinant

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dihydrotanshinone I preconditions myocardium against ischemic injury via PKM2 glutathionylation sensitive to ROS

doi: 10.1016/j.apsb.2022.07.006

Figure Lengend Snippet: Characterization of the PKM2 glutathionylation amino acid sites. Immunoblot analysis of nuclear PKM2 in HEK-293T cells (A) and quantitative results (B) in HEK-293T cells pre-treated with diamide or H 2 O 2 for 4 h ( n = 3). (C) A scheme showing how the DCP-Bio1 probe detects the oxidation of Cys423/424 in PKM2. (D) Immunoblot analysis of PKM2 protein level pulled out by DCP-Bio1 probe in HEK 293T cells treated with H 2 O 2 , diamide or DT for 4 h ( n = 3). (E) Quantitative analysis of the PKM2 protein level pulled out by DCP-Bio1 probe ( n = 3). (F) IP analysis of Flag-PKM2 and mutants. (G) LC–MS/MS analysis of PKM2 glutathionylation in HL-1 cells. The amino acid site Cys423 and Cys424 were modified by GSSG (data from Project PXD012171). (H) A structural model of GSSG binding to PKM2. GSSG is docked to the disulfide bond of Cys423 and Cys424. The hydrogen bonds formed at G501, K504 and R447 between GSSG and PKM2.

Article Snippet: Anti-PKM2 antibody (cat#4053), anti-Lamin A/C antibody (cat# 2032) and anti-HIF-1 α antibody (cat#36169) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Liquid Chromatography with Mass Spectroscopy, Modification, Binding Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Dihydrotanshinone I preconditions myocardium against ischemic injury via PKM2 glutathionylation sensitive to ROS

doi: 10.1016/j.apsb.2022.07.006

Figure Lengend Snippet: The preconditioning effect of DT in mice. (A) IP analysis of the binding of α -GSH to PKM2 and quantitative results ( n = 3). (B) Immunoblot analysis of HIF-1 α and quantitative results in the nuclear fractions of mouse heart ( n = 3). (C) mRNA level of Pdk1 , Hmox1 , Vegfa and Il10 in the heart ( n = 6). Myocardial infarction in mice after coronary ligation without reperfusion was examined three days after ligation. (D) Representative images and calculated infarct size of the myocardium (Scale bar: 2 mm, n = 6). The effects on myocardial injury, fibrosis and ventricular function were examined 3 weeks after the surgery. (E) Masson staining of heart sections and quantitative fibrotic area (Scale bar: 50 μm, n = 6). (F) Ejection fraction (EF) ( n = 6). (G) Fractional shortening (FS) ( n = 6). The results are expressed as the means and SEM. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, statistically not significant.

Article Snippet: Anti-PKM2 antibody (cat#4053), anti-Lamin A/C antibody (cat# 2032) and anti-HIF-1 α antibody (cat#36169) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Binding Assay, Western Blot, Ligation, Staining

Journal: PLoS ONE

Article Title: Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome

doi: 10.1371/journal.pone.0151635

Figure Lengend Snippet: Correlation of PKM2 and LDHA expression with clinicopathological factors (UCLH cohort).

Article Snippet: The sections were then incubated with primary antibody for one hour at room temperature for CD8 (CD8A Rabbit polyclonal antibody, Abnova, UK, Cat# PAB11235, 1:200) and Ki-67 (Rabbit polyclonal to Ki-67, Abcam, UK, Cat# ab15580, 1:300), and overnight at 4°C for PKM2 (Monoclonal mouse anti-human and rat PKM2, ScheBo®Biotech, Giessen, Germany, Cat# S-1, 1:100) and LDHA (LDHA/LDHC (C28H7) Rabbit Monoclonal antibody, Cell Signaling, UK, Cat #3558, 1:250) in PBS.

Techniques: Expressing, Biomarker Discovery

Journal: PLoS ONE

Article Title: Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome

doi: 10.1371/journal.pone.0151635

Figure Lengend Snippet: (A) Well differentiated area of tumour showing weak PKM2 expression (red arrow), with high expression in poorly differentiated areas (brown colour, yellow arrow) (x100 magnification). (B) Growing margin of tumour nodules with strong expression of PKM2 (red arrow) (x100 magnification). (C) Membranous expression of PKM2 (red arrow) (x200 magnification). (D) Heterogeneous expression of PKM2 with predominant expression in the proliferating cells (red arrow) (x200 magnification). (E) Strongly positive tumour expression of PKM2 in vascular invasion (red arrow) (x100 magnification). (F) Strongly positive tumour expression of PKM2 with muscular invasion (red arrow) (x100 magnification).

Article Snippet: The sections were then incubated with primary antibody for one hour at room temperature for CD8 (CD8A Rabbit polyclonal antibody, Abnova, UK, Cat# PAB11235, 1:200) and Ki-67 (Rabbit polyclonal to Ki-67, Abcam, UK, Cat# ab15580, 1:300), and overnight at 4°C for PKM2 (Monoclonal mouse anti-human and rat PKM2, ScheBo®Biotech, Giessen, Germany, Cat# S-1, 1:100) and LDHA (LDHA/LDHC (C28H7) Rabbit Monoclonal antibody, Cell Signaling, UK, Cat #3558, 1:250) in PBS.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome

doi: 10.1371/journal.pone.0151635

Figure Lengend Snippet: Immunostaining of Miapaca-2 cells with PKM2 (A) and LDHA (B) is shown in the lower panel. Strong cytoplasmic and nuclear staining is noted in proliferating cells.

Article Snippet: The sections were then incubated with primary antibody for one hour at room temperature for CD8 (CD8A Rabbit polyclonal antibody, Abnova, UK, Cat# PAB11235, 1:200) and Ki-67 (Rabbit polyclonal to Ki-67, Abcam, UK, Cat# ab15580, 1:300), and overnight at 4°C for PKM2 (Monoclonal mouse anti-human and rat PKM2, ScheBo®Biotech, Giessen, Germany, Cat# S-1, 1:100) and LDHA (LDHA/LDHC (C28H7) Rabbit Monoclonal antibody, Cell Signaling, UK, Cat #3558, 1:250) in PBS.

Techniques: Immunostaining, Staining

Journal: PLoS ONE

Article Title: Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome

doi: 10.1371/journal.pone.0151635

Figure Lengend Snippet: PKM2 was strongly expressed by pancreatic cancer tissue specimens and was significantly higher than in normal, pancreatitis, pancreatic cyst and PanIN tissues (P<0.001). Expression of LDHA was significantly higher in pancreatic cancer than in normal pancreas (P<0.001), whereas, there was no significant differences in LDHA expression between pancreatic cancer, PanIN, pancreatic cysts and pancreatitis.

Article Snippet: The sections were then incubated with primary antibody for one hour at room temperature for CD8 (CD8A Rabbit polyclonal antibody, Abnova, UK, Cat# PAB11235, 1:200) and Ki-67 (Rabbit polyclonal to Ki-67, Abcam, UK, Cat# ab15580, 1:300), and overnight at 4°C for PKM2 (Monoclonal mouse anti-human and rat PKM2, ScheBo®Biotech, Giessen, Germany, Cat# S-1, 1:100) and LDHA (LDHA/LDHC (C28H7) Rabbit Monoclonal antibody, Cell Signaling, UK, Cat #3558, 1:250) in PBS.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome

doi: 10.1371/journal.pone.0151635

Figure Lengend Snippet: (A) Well differentiated tumours with negative PKM2 expression had strong infiltration by CD8+ positive T-lymphocytes (x200 magnification). (B) Poorly differentiated tumours strongly positive for PKM2 had sparse infiltration by CD8+ positive T-lymphocytes (x200 magnification). There was a significant negative correlation between CD8+ positive cells and both PKM2 (C) & (D) and LDHA staining (E) & (F).

Article Snippet: The sections were then incubated with primary antibody for one hour at room temperature for CD8 (CD8A Rabbit polyclonal antibody, Abnova, UK, Cat# PAB11235, 1:200) and Ki-67 (Rabbit polyclonal to Ki-67, Abcam, UK, Cat# ab15580, 1:300), and overnight at 4°C for PKM2 (Monoclonal mouse anti-human and rat PKM2, ScheBo®Biotech, Giessen, Germany, Cat# S-1, 1:100) and LDHA (LDHA/LDHC (C28H7) Rabbit Monoclonal antibody, Cell Signaling, UK, Cat #3558, 1:250) in PBS.

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome

doi: 10.1371/journal.pone.0151635

Figure Lengend Snippet: (A) Tumours with strongly positive PKM2 expression had most of the nucleus stained for Ki-67 (x100 magnification). (B) Tumours weakly positive for PKM2 had scant Ki67 staining (x100 magnification). (C) & (D) Correlation between PKM2 staining and Ki-67 positive cells. (E) & (F) Correlation between LDHA staining and Ki-67 positive cells. There was a significant correlation for both PKM2 and LDHA.

Article Snippet: The sections were then incubated with primary antibody for one hour at room temperature for CD8 (CD8A Rabbit polyclonal antibody, Abnova, UK, Cat# PAB11235, 1:200) and Ki-67 (Rabbit polyclonal to Ki-67, Abcam, UK, Cat# ab15580, 1:300), and overnight at 4°C for PKM2 (Monoclonal mouse anti-human and rat PKM2, ScheBo®Biotech, Giessen, Germany, Cat# S-1, 1:100) and LDHA (LDHA/LDHC (C28H7) Rabbit Monoclonal antibody, Cell Signaling, UK, Cat #3558, 1:250) in PBS.

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome

doi: 10.1371/journal.pone.0151635

Figure Lengend Snippet: Both PKM2 (A) and LDHA (B) had a significant prognostic impact on patient survival (p = 0.016, 0.029, log rank test, respectively) and the combined expression for both markers further stratified the patients (C) (p = 0.003, log rank test). As expected, patients who underwent surgical resection had a longer survival than unresected patients.

Article Snippet: The sections were then incubated with primary antibody for one hour at room temperature for CD8 (CD8A Rabbit polyclonal antibody, Abnova, UK, Cat# PAB11235, 1:200) and Ki-67 (Rabbit polyclonal to Ki-67, Abcam, UK, Cat# ab15580, 1:300), and overnight at 4°C for PKM2 (Monoclonal mouse anti-human and rat PKM2, ScheBo®Biotech, Giessen, Germany, Cat# S-1, 1:100) and LDHA (LDHA/LDHC (C28H7) Rabbit Monoclonal antibody, Cell Signaling, UK, Cat #3558, 1:250) in PBS.

Techniques: Expressing