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

Journal: Frontiers in Immunology

Article Title: The Circadian Clock Protein BMAL1 Acts as a Metabolic Sensor In Macrophages to Control the Production of Pro IL-1β

doi: 10.3389/fimmu.2021.700431

Figure Lengend Snippet: Mitochondrial respiration and Krebs cycle glucose flux is altered in Bmal1 -/- macrophages. Bmal1 +/+ and Bmal1 -/- BMDMs were stimulated with LPS (100 ng/ml), subjected to a Seahorse XF mitochondrial stress test. (A) OCR of BMDMs was measured, and the metabolic trace illustrates changes in OCR following injection of the mitochondrial stress test compounds oligomycin, FCCP, and rotenone/antimycin a. (B) Measures of basal respiration, ATP production, maximal respiration, and proton leak were derived from (A) . Assay results are presented +/- SEM and are representative of n=3 independent experiments. BMDMs were isolated, seeded in 10 mM U-13C6 glucose, and stimulated with LPS for 8 hours. BMDMs were lysed and metabolites were quenched and measured via GC-MS to trace Krebs cycle flux of labelled glucose. (C) A schematic of U- 13 C 6 glucose-derived carbon Krebs cycle flux and incorporation into metabolic intermediates. Relative abundance of U- 13 C 6 -labelled (D) pyruvate, (F) citrate, (J) itaconate, (K) α-Ketoglutarate, and (M) succinate, and mass isotopologue distribution (MID) of (E) pyruvate, (G) citrate, and (L) α-Ketoglutarate, and (N) succinate were measured. MID values of low abundance isotopologues are excluded for clarity. (I) Ratio of m+2 citrate/m+3 pyruvate representative of U- 13 C 6 glucose-derived carbon flux through pyruvate dehydrogenase. Data presented is n=4 +/- SEM. (H) BMDMs were stimulated with LPS and protein expression of PDH was analysed by Western blot using β-Actin as a loading control. Densitometry is relative to the Bmal1 +/+ control band. This band is indicated by a * symbol. Data presented is representative of n=3 independent experiments. Statistical analysis was performed for Seahorse XF data and U- 13 C 6 relative abundance values by one-way ANOVA with Sidak’s multiple comparisons test and for MID values by multiple student’s t-tests with Holm-Sidak correction for multiple comparisons (*p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001).

Article Snippet: Nitrocellulose or PVDF membranes were probed with antibodies for BMAL1 (14020S, CST), TOM20 (sc-11415, Santa Cruz), β-Actin (4967S, CST) Pro-IL1β (AF-401-NA, CST), Complex II WB Antibody Cocktail (ab110410, Abcam) HIF-1α (14179, CST), GLUT1 (12939, CST), PDHα (3205S, CST), PKM2 (4053, CST), α-Tubulin (3873, CST), Histone H3 (4499, CST), and pSTAT3-Tyr705 (9145, CST).

Techniques: Injection, Derivative Assay, Isolation, Gas Chromatography-Mass Spectrometry, Expressing, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: The Circadian Clock Protein BMAL1 Acts as a Metabolic Sensor In Macrophages to Control the Production of Pro IL-1β

doi: 10.3389/fimmu.2021.700431

Figure Lengend Snippet: Schematic of immunometabolic changes in macrophages with deletion of Bmal1. Glucose metabolism is increased in macrophages with deletion of Bmal1 which potentiates expression of the pro-inflammatory cytokine IL-1β. In the absence of Bmal1 in macrophages, increased expression of the glucose transporter GLUT1 leads to increased glucose uptake and higher glycolytic pathway activity. Increased dimerization of the glycolytic enzyme PKM2 facilitates its translocation to the nucleus where it phosphorylates STAT3 to drive IL-1β expression. In the mitochondria, flux of pyruvate through pyruvate dehydrogenase (PDH) and oxygen consumption is increased alongside increased Krebs cycle flux which fuels accumulation of the intermediate succinate. Activity of the electron transport chain complex succinate dehydrogenase (SDH) is also increased in Bmal1 -/- macrophages which produces heightened levels of ROS which stabilizes HIF-1α to also promote IL-1β expression. Therefore, BMAL1 is regulating glucose metabolism in macrophages to impact upon the expression of IL-1β.

Article Snippet: Nitrocellulose or PVDF membranes were probed with antibodies for BMAL1 (14020S, CST), TOM20 (sc-11415, Santa Cruz), β-Actin (4967S, CST) Pro-IL1β (AF-401-NA, CST), Complex II WB Antibody Cocktail (ab110410, Abcam) HIF-1α (14179, CST), GLUT1 (12939, CST), PDHα (3205S, CST), PKM2 (4053, CST), α-Tubulin (3873, CST), Histone H3 (4499, CST), and pSTAT3-Tyr705 (9145, CST).

Techniques: Expressing, Activity Assay, Translocation Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Effect of Huaier on the proliferation and apoptosis of human gastric cancer cells through modulation of the PI3K/AKT signaling pathway

doi: 10.3892/etm.2015.2600

Figure Lengend Snippet: Effect of Huaier extract on the protein expression of AKT1, PI3K, PTEN, PDK1, caspase-9 and Bcl-2 in SGC7901 cells. The expression of GAPDH was used as an internal control. The SGC7901 cells were treated with 0, 2.5 and 5 mg/ml Huaier for 24 h, and (A) western blotting was used to analyze the protein expression. (B) Quantification of the data. The experiment was performed in triplicate, and the data are expressed as the mean ± standard deviation of the three separate experiments. *P<0.05 and **P<0.01, compared with the control. p-, phosphorylated-; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase; PDK1, pyruvate dehydrogenase kinase isoform 1; Bcl-2, B-cell lymphoma 2; BAD, Bcl-2-associated death promoter.

Article Snippet: Primary monoclonal antibodies against AKT (1:1,000; 10176-2-AP), PDK1 (1:1,000; 10026-1-AP), Bcl-2-associated death promoter (BAD; 1:1,000; 10435-1-AP) and GAPDH (1:4,000; 60004-1-Ig) were purchased from Proteintech (Manchester, UK), while monoclonal antibodies against p-AKT (The-308; 1:800; 2118-1), p-AKT (Ser-473; 1:800; 2214-1), PI3K (1:1,000; 1593-S), PTEN (1:1,000; 5171-1), p-PTEN (1:1,000; 2134-1), Bcl-2 (1:1,000; 10026-1-AP), pro-caspase-9 (1:1,000; 1023-1) and cyclin B1 (1:2,000; 1495-1) were obtained from Abcam (Cambridge, UK), and monoclonal anti-cleaved-caspase-9 (1:1,000; sc-22182) was from Santa Cruz Biotechnology, Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Effect of Huaier on the proliferation and apoptosis of human gastric cancer cells through modulation of the PI3K/AKT signaling pathway

doi: 10.3892/etm.2015.2600

Figure Lengend Snippet: Effect of Huaier extract on the protein expression of AKT1, PI3K, PTEN, PDK1, caspase-9 and Bcl-2. The expression of GAPDH was used as an internal control. MKN45 cells were treated with 0, 2.5 and 5 mg/ml for 24 h, and (A) western blotting was used to analyze the protein expression. (B) Quantification of the data. The experiment was performed in triplicate, and the data are expressed as the mean ± standard deviation of the three separate experiments. *P<0.05 and **P<0.01, compared with the control. p-, phosphorylated-; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase; PDK1, pyruvate dehydrogenase kinase isoform 1; Bcl-2, B-cell lymphoma 2; BAD, Bcl-2-associated death promoter.

Article Snippet: Primary monoclonal antibodies against AKT (1:1,000; 10176-2-AP), PDK1 (1:1,000; 10026-1-AP), Bcl-2-associated death promoter (BAD; 1:1,000; 10435-1-AP) and GAPDH (1:4,000; 60004-1-Ig) were purchased from Proteintech (Manchester, UK), while monoclonal antibodies against p-AKT (The-308; 1:800; 2118-1), p-AKT (Ser-473; 1:800; 2214-1), PI3K (1:1,000; 1593-S), PTEN (1:1,000; 5171-1), p-PTEN (1:1,000; 2134-1), Bcl-2 (1:1,000; 10026-1-AP), pro-caspase-9 (1:1,000; 1023-1) and cyclin B1 (1:2,000; 1495-1) were obtained from Abcam (Cambridge, UK), and monoclonal anti-cleaved-caspase-9 (1:1,000; sc-22182) was from Santa Cruz Biotechnology, Inc. (Shanghai, China).

Techniques: Expressing, Western Blot, Standard Deviation