Journal: Frontiers in oncology

Article Title: The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

doi: 10.3389/fonc.2021.644492

Figure Lengend Snippet: FIGURE 3 | P7C3 reduces the protein level of PGK1 and PGK2. (A) Representative spots of P7C3 target proteins, including the glycolytic process. (B) Western blotting for detecting protein levels of P7C3 target proteins that are related to glycolysis after U87MG and U118MG cells were treated with P7C3 at a concentration of 0 mM, 30 mM, and 50 mM for 24 h. (C) PGK1 activity levels in P7C3-treated U87MG and U118MG cells at a concentration of 0 mM, 30 mM, and 50 mM (n = 4). (D) The expression profiles of PGK1 and PGK2 mRNA levels. Data were obtained from the Human Protein Atlas database. The images are available at: https://www. proteinatlas.org/ENSG00000102144-PGK1/cell, and (https://www.proteinatlas.rg/ENSG00000170950-PGK2/cell). (E) Statistical analysis of PGK1 mRNA level among WHO grade II-IV glioma. The data were based on TCGA and CGGA and are available at: http://gliovis.bioinfo.cnio.es/. (F) Kaplan–Meier survival analysis of PGK1 mRNA level in glioma patients. The data were based on TCGA and CGGA and are available at: http://gliovis.bioinfo.cnio.es/. (The data were expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Recombinant human PGK1 (GST-PGK1, ag12119, Proteintech, USA) was purchased from Proteinteck Group, Inc. Antibodies used in this study are as follows: HK1 (#2024, CST, USA), HK2 (#2867, CST, USA), GCK (#3782, CST, USA), ALDOA (#8060, CST, USA), PGK1(sc-130335, Santa Cruz, USA), PGK2 (ab183031, Abcam, USA), GST (#2624, CST, USA), GAPDH (#5174, CST, USA), Beclin-1 (#3495, CST, USA), LC3A/B (#12741, CST, USA), Iba1 (ab178846, Abcam, USA), SOX10 (ab227680, Abcam, USA), GFAP (ab68428, Abcam, USA), and b-Tubulin (#2128, CST, USA).

Techniques: Western Blot, Concentration Assay, Activity Assay, Expressing

Journal: Frontiers in oncology

Article Title: The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

doi: 10.3389/fonc.2021.644492

Figure Lengend Snippet: FIGURE 4 | P7C3 directly binds to PGK1. (A) Streptavidin affinity assay for P7C3-Bio binds to PGK1 in P7C3-treated glioma cells. Cells in culture were treated with 50 mM P7C3-Bio for 24 h, and then streptavidin agarose was added to incubate with the cell lysates. (B) Streptavidin affinity assay for P7C3-Bio binds to PGK1 upon cell lysates. Cell lysates were incubated with 10 mM P7C3-Bio at 37°C for 1 h, and then streptavidin agarose was added to incubate with the cell lysates. (C) Streptavidin affinity assay for P7C3-Bio binds to purified PGK1 protein. In total, 25 mg recombinant human PGK1 solution was incubated with 10 mM P7C3-Bio at 37°C for 1 h, and then streptavidin agarose was added to incubate with the cell lysates. (D) Motif analysis based on top 100 P7C3 target proteins was performed by MEME, and three motifs were identified. The motif consensus and motif locations at PGK1 are shown. (E) The P7C3 binding sites of PGK1 were identified by ESI-LC-MSMS. The P7C3 binding sites were identified by the predicted peptide mass plus P7C3 [C21H18Br2N2O with a loss of a water] and minus one H.

Article Snippet: Recombinant human PGK1 (GST-PGK1, ag12119, Proteintech, USA) was purchased from Proteinteck Group, Inc. Antibodies used in this study are as follows: HK1 (#2024, CST, USA), HK2 (#2867, CST, USA), GCK (#3782, CST, USA), ALDOA (#8060, CST, USA), PGK1(sc-130335, Santa Cruz, USA), PGK2 (ab183031, Abcam, USA), GST (#2624, CST, USA), GAPDH (#5174, CST, USA), Beclin-1 (#3495, CST, USA), LC3A/B (#12741, CST, USA), Iba1 (ab178846, Abcam, USA), SOX10 (ab227680, Abcam, USA), GFAP (ab68428, Abcam, USA), and b-Tubulin (#2128, CST, USA).

Techniques: Incubation, Recombinant, Binding Assay

Journal: Frontiers in oncology

Article Title: The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

doi: 10.3389/fonc.2021.644492

Figure Lengend Snippet: FIGURE 5 | P7C3 accelerates PGK1 protein degradation. (A) PGK1 mRNA level was measured by RT-PCR. P7C3 upregulates the PGK1 mRNA levels in U87MG and U118MG cells (n = 3). (B) Time-course assay of P7C3 effects on PGK1 degradation. U87MG and U118MG cells were treated by CHX (100 ug/mL) with or without P7C3 (50 mM) for 24 h, and then PGK1 protein levels were detected by western blotting. (C) Protein level curve for PGK1 based on time-course analysis of U87MG and U118MG cells (n = 3). (D) Western blotting of PGK1 protein degradation pathway. MG132 (5 mM) and CQ (20 mM) were used to treat cells for 6 h, followed by P7C3 treatment for another 18 h, and the protein level of PGK1 was detected by western blotting. (E) The statistical analysis of the quantized protein levels of PGK1 in U87MG and U118MG cells (n = 3). (F) Transmission electron microscopy assay after glioma cells were treated with P7C3 for 24 hours. Autophagosomes, autolysosomes, and phagophores are indicated by yellow, green, and red arrows (scale bar: original mage, 5 mm; Zoom image, 1 mm). (G) Western blotting for two key autophagy-associated proteins Beclin-1, and LC3A/B, after U87MG and U118MG cells were treated with 0 mM, 30 mM, and 50 mM P7C3 for 24 h. (Data are expressed as mean ± SD, nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns means none significance).

Article Snippet: Recombinant human PGK1 (GST-PGK1, ag12119, Proteintech, USA) was purchased from Proteinteck Group, Inc. Antibodies used in this study are as follows: HK1 (#2024, CST, USA), HK2 (#2867, CST, USA), GCK (#3782, CST, USA), ALDOA (#8060, CST, USA), PGK1(sc-130335, Santa Cruz, USA), PGK2 (ab183031, Abcam, USA), GST (#2624, CST, USA), GAPDH (#5174, CST, USA), Beclin-1 (#3495, CST, USA), LC3A/B (#12741, CST, USA), Iba1 (ab178846, Abcam, USA), SOX10 (ab227680, Abcam, USA), GFAP (ab68428, Abcam, USA), and b-Tubulin (#2128, CST, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transmission Assay, Electron Microscopy

Journal: Frontiers in oncology

Article Title: The Neurogenic Compound P7C3 Regulates the Aerobic Glycolysis by Targeting Phosphoglycerate Kinase 1 in Glioma.

doi: 10.3389/fonc.2021.644492

Figure Lengend Snippet: FIGURE 7 | P7C3 inhibits glioma in vivo. (A) Representative in vivo IVIS Luc fluorescence images of xenografts mice on days 7 and 21 after injection of U87MG cells. (B) Statistical result of photon flux in xenografts mice on days 7 and 21 (n = 12). (C) Kaplan–Meier curves of survival rate of xenografts mice (n = 6). (D) Immunohistochemical staining of PGK1 in the indicated tumor tissues was performed, PGK1 positive cells were stained brown (scale bar: 20 mm). (E) Immunohistochemical staining of Ki67 in the indicated tumor tissues was performed. Ki67 positive cells were stained brown (scale bar: 50 mm). (F) TUNEL analyses of the indicated tumor tissues were performed. Apoptotic cells were stained brown (scale bar: 50 mm). (G) Immunohistochemical staining of labeling astrocytes, microglia, and oligodendrocytes in the mice brain by using GFAP, Iba1, and SOX10, respectively (scale bar: 100 mm). (H) Statistical analysis of GFAP-positive, Iba1- positive, and SOX10-positive cells in the mouse brain (n = 3). (The data were expressed as mean ± SD, nsP > 0.05, *P < 0.05, ns means none significance).

Article Snippet: Recombinant human PGK1 (GST-PGK1, ag12119, Proteintech, USA) was purchased from Proteinteck Group, Inc. Antibodies used in this study are as follows: HK1 (#2024, CST, USA), HK2 (#2867, CST, USA), GCK (#3782, CST, USA), ALDOA (#8060, CST, USA), PGK1(sc-130335, Santa Cruz, USA), PGK2 (ab183031, Abcam, USA), GST (#2624, CST, USA), GAPDH (#5174, CST, USA), Beclin-1 (#3495, CST, USA), LC3A/B (#12741, CST, USA), Iba1 (ab178846, Abcam, USA), SOX10 (ab227680, Abcam, USA), GFAP (ab68428, Abcam, USA), and b-Tubulin (#2128, CST, USA).

Techniques: In Vivo, Injection, Immunohistochemical staining, Staining, TUNEL Assay, Labeling

Journal: PLoS ONE

Article Title: Allergen-Induced Dermatitis Causes Alterations in Cutaneous Retinoid-Mediated Signaling in Mice

doi: 10.1371/journal.pone.0071244

Figure Lengend Snippet: Systemic and topical OVA sensitization results in inflammation, disturbs epidermal barrier homeostasis, and induces PPARδ target gene expression in skin.

Article Snippet: FABP5 protein levels were determined in protein lysates prepared from whole mouse skin according to the protocol indicated in using the rabbit FABP5 polyclonal antibody purchased from ProteinTech (Chicago, IL).

Techniques: Targeted Gene Expression, Binding Assay

Journal: PLoS ONE

Article Title: Allergen-Induced Dermatitis Causes Alterations in Cutaneous Retinoid-Mediated Signaling in Mice

doi: 10.1371/journal.pone.0071244

Figure Lengend Snippet: ( a ) IL-4 serum levels after systemic with or without additional topical OVA sensitization (n = 8). ( b ) ATRA levels in mouse skin determined by HPLC MS-MS method upon systemic (i.p.) and systemic plus topical (i.p.+e.c.) OVA sensitization (n = 3/group). ( c ) Ratio of Fabp5 vs. Crabp2 expression in the skin of OVA-treated mice (n = 6/group) compared to control mice (PBS i.p.). Data are presented as mean values ± SEM. Statistical significance ( p ) is based on one-way ANOVA followed by Tukey’s multiple comparison test for gene expression results and ELISA data. For HPLC MS-MS results, significance was determined using Student’s t -test.

Article Snippet: FABP5 protein levels were determined in protein lysates prepared from whole mouse skin according to the protocol indicated in using the rabbit FABP5 polyclonal antibody purchased from ProteinTech (Chicago, IL).

Techniques: Tandem Mass Spectroscopy, Expressing, Control, Comparison, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Allergen-Induced Dermatitis Causes Alterations in Cutaneous Retinoid-Mediated Signaling in Mice

doi: 10.1371/journal.pone.0071244

Figure Lengend Snippet: ( a ) Fabp5 protein levels in the skin of mice with allergen-induced dermatitis. 150 µg proteins were loaded per lane and beta-actin was used as control for even protein loading. ( b ) Immunohistochemical analysis of Fabp5 protein expression in five-micrometer back skin sections of OVA-sensitized mice. ( c ) ATRA-induced nuclear receptor-mediated signaling pathways depending on the predominant cellular transport protein.

Article Snippet: FABP5 protein levels were determined in protein lysates prepared from whole mouse skin according to the protocol indicated in using the rabbit FABP5 polyclonal antibody purchased from ProteinTech (Chicago, IL).

Techniques: Control, Immunohistochemical staining, Expressing, Protein-Protein interactions