Journal: Acta pharmaceutica Sinica. B

Article Title: Protocatechuic aldehyde protects cardiomycoytes against ischemic injury via regulation of nuclear pyruvate kinase M2.

doi: 10.1016/j.apsb.2021.03.021

Figure Lengend Snippet: Figure 2 Identification of PKM2 as a direct binding target for PCA. (A) The employed drug affinity responsive target stabilization (DARTS) assay detects a marked increase in w60 kD band upon PCA incubation in pronase digested H9C2 cell lysates. (B) Immunoblot analysis of PKM2 in pronase-digested cell lysate (n Z 3). (C) Recombinant PKM2 in pronase-digested H9C2 cell lysates. PKM2 degradation in H9C2 cell lysates (D) and in intact cells (E) (n Z 3). (F) Surface plasmon resonance (SPR) binding curve fit to a Kd Z 5.76 nmol/L for PCA and PKM2 (n Z 3). Results are shown as mean SD; *P < 0.05, ***P < 0.001.

Article Snippet: Antibodies against GAPDH (60004-1-Ig), b-catenin (51067-2-AP, 66379-1-Ig), PCNA (10205-2-AP), caspase 3 (66470-2-Ig), BAX (60267-1-Ig), BCL-2 (60178-1-Ig), PKM2 (60268-1-Ig), b-tubulin (10068-1-AP) and TCF4 (22337-1-AP) were obtained from Proteintech (Wuhan, China).

Techniques: Binding Assay, Incubation, Western Blot, Recombinant, SPR Assay

Journal: Acta pharmaceutica Sinica. B

Article Title: Protocatechuic aldehyde protects cardiomycoytes against ischemic injury via regulation of nuclear pyruvate kinase M2.

doi: 10.1016/j.apsb.2021.03.021

Figure Lengend Snippet: Figure 3 PCA PKM2-dependently protects cardiomyocytes. Primary neonatal rat ventricular myocytes (NRVMs) were cultured in glucose-free DMEM under 1% O2 (OGD) for 4 h in the presence of protocatechuic aldehyde (PCA). (A) Cell survival in NRVMs exposed to OGD for 6 h (n Z 6). (B) Intracellular ROS production (n Z 6). (C) Representative images of mitochondrial permeability transition pore (mPTP) and quantification of relative fluorescence intensity (n Z 6, one of three independent experiments). Scale bar: 10 mm. (D) Mitochondrial fission was detected by Mito-Tracker Red (one of 3 independent experiments) and quantification analysis. Scale bar: 10 mm. (E) Caspase 3 activity in NRVMs (n Z 6). (F) Representative Western blots of BAX, BCL-2, caspase 3, cleaved caspase 3 (c-caspase 3) and the ratio of BAX/BCL-2 (n Z 3). (G) Representative images of TUNEL staining and quantification analysis the percentage of apoptosis cells. Scale bars, 100 mm (n Z 3). (H) Data of Annexin V/PI double staining flow cytometry; the percentage for each panel indicates the percentage of apoptotic cells and quantification of the percentage of apoptotic cells (n Z 3). Results are shown as mean SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies against GAPDH (60004-1-Ig), b-catenin (51067-2-AP, 66379-1-Ig), PCNA (10205-2-AP), caspase 3 (66470-2-Ig), BAX (60267-1-Ig), BCL-2 (60178-1-Ig), PKM2 (60268-1-Ig), b-tubulin (10068-1-AP) and TCF4 (22337-1-AP) were obtained from Proteintech (Wuhan, China).

Techniques: Cell Culture, Permeability, Activity Assay, Western Blot, TUNEL Assay, Staining, Double Staining, Cytometry

Journal: Acta pharmaceutica Sinica. B

Article Title: Protocatechuic aldehyde protects cardiomycoytes against ischemic injury via regulation of nuclear pyruvate kinase M2.

doi: 10.1016/j.apsb.2021.03.021

Figure Lengend Snippet: Figure 4 PCA promotes the nuclear localization of PKM2. Primary neonatal rat ventricular myocytes (NRVMs) were exposed to 1% O2 in glucose-free DMEM (OGD) for 4 h in the presence of protocatechuic aldehyde (PCA). (A) PKM2 activity in NRVMs (n Z 6). (B) Immuno- fluorescence image of endogenous PKM2 (one of three independent experiments. Scale bar: 10 mm. (C) Nuclear PKM2 protein expression (n Z 3). (D) Cytoplasmic PKM2 protein expression (n Z 3). (E) Total PKM2 protein expression (n Z 3). (F) Docking analysis illustrates the interaction between PCA and PKM2. The residues that are likely to participate in the interactions with PCA are labeled. (G) Representative co- immunoprecipitation (co-IP) analysis of PKM2 and SIRT6 in the NRVMs (n Z 3). (H) Representative co-IP of PKM2 and b-catenin in the NRVMs (n Z 3). Results are shown as mean SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies against GAPDH (60004-1-Ig), b-catenin (51067-2-AP, 66379-1-Ig), PCNA (10205-2-AP), caspase 3 (66470-2-Ig), BAX (60267-1-Ig), BCL-2 (60178-1-Ig), PKM2 (60268-1-Ig), b-tubulin (10068-1-AP) and TCF4 (22337-1-AP) were obtained from Proteintech (Wuhan, China).

Techniques: Activity Assay, Expressing, Labeling, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Acta pharmaceutica Sinica. B

Article Title: Protocatechuic aldehyde protects cardiomycoytes against ischemic injury via regulation of nuclear pyruvate kinase M2.

doi: 10.1016/j.apsb.2021.03.021

Figure Lengend Snippet: Figure 6 The myocardial protection effect of PCA is dependent on PKM2. Mice were orally administrated with protocatechuic aldehyde (PCA) for 3 weeks after coronary artery ligation. (A) Representative photomicrographs for HE staining of cardiac tissue sections (n Z 6), Scale bar, 50 mm. (B) Representative photomicrographs for masson staining of cardiac tissue sections (n Z 6). Scale bar, 50 mm. (C) 4-HNE contents in the heart (n Z 6). (D) Representative M mode images of echocardiography from the assayed groups under treatments as indicated (n Z 6). (E) Ejection fractions (EF) and shortening fraction (FS) in mice (n Z 6). (F) Representative co-IP analysis of PKM2 and b-catenin in the heart (n Z 3). The mRNA levels of Myc (G), Ccnd1 (H) and Sgk1 (I) in the heart (n Z 6). (J) Immunocytochemical staining of BAX, BCL-2 and c- caspase 3 protein expression (n Z 6), scale bar Z 50 mm. (K) Representative TUNEL staining images and quantification of TUNEL positive cells (n Z 6). Results are shown as mean SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies against GAPDH (60004-1-Ig), b-catenin (51067-2-AP, 66379-1-Ig), PCNA (10205-2-AP), caspase 3 (66470-2-Ig), BAX (60267-1-Ig), BCL-2 (60178-1-Ig), PKM2 (60268-1-Ig), b-tubulin (10068-1-AP) and TCF4 (22337-1-AP) were obtained from Proteintech (Wuhan, China).

Techniques: Ligation, Staining, Co-Immunoprecipitation Assay, Expressing, TUNEL Assay