Journal: Scientific reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation.

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Fig. 2. Diaphragm cell type identification. a Clustering algorithm with a resolution of 0.6 identified 15 clusters. b Dot plots of key marker gene expression levels across cell clusters. The average expression (dot colour intensity) and cell percentage (dot size) of the two top marker genes for each cluster were compared. c. Violin plots showing the relative expression levels of selected genes for different clusters. Cells are coloured according to the expression of the specific marker genes. UMAP of snRNA-seq data displayed the presence of Fibroblast 1 containing Cluster 1, 2, 8 marked by Pfkfb3 and Tbc1d1 (d); Myocyte 1 corresponding to Cluster 5 marked by Myh7b and Mylk3 (e); Myocyte 2 corresponding to Cluster 7 marked by Myh1 and Myl1 (f); Fibroblast 2 corresponding to Cluster 6 marked by Col15a1 and Pdgfrα (g); Endothelial cell including Cluster 9,14 marked by Tek and Emcn (h); Neurocyte corresponding to Cluster 12 marked by Pak5 and Ntng1 (i); Immune cell corresponding to Cluster 13 marked by Ikzf1 and Cd163 (j); Satellite cell corresponding to Cluster 13 marked by Pax7 and Fgfr4 (k); Pericytes and smooth muscle cell corresponding to Cluster 15 marked by Acta2 and Rgs5 (l).

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Marker, Gene Expression, Expressing

Journal: Scientific reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation.

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Fig. 4. Differentially expressed genes and Kyoto Encyclopedia of Genes and Genomes analysis. Volcano plots showing the top twenty up- or downregulated genes for each cell type. The red dots represent upregulated genes, and the blue dots represent downregulated genes. (P value < 0.05, and |log2foldchange| > 0.58). Bubble plot indicating the top enriched pathways for each cell type based on KEGGpathway enrichment analysis of differentially expressed genes(www.kegg.jp/kegg/kegg1.html). The sizes of the dots represent the number of genes included in each pathway. The colour gradient of dots represents the adjusted P values of each enriched pathway. The genes Pfkfb3, Tbc1d1 and the insulin signaling pathway; Pdgfd and the PI3K-Akt signaling pathway; Cxcr2 and PLD, Rap1 signaling pathways; Ccl21 and chemical carcinogenesis-reactive oxygen species signaling pathway; Mef2c and the calcium signaling pathway; Negr1 and leukocyte transendothelial migration signaling pathway are labelled by the black and red bars in Panels a, b, c, d, e, and f.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Protein-Protein interactions, Migration

Journal: Scientific reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation.

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Fig. 6. Protein-protein interaction network (PPI network).We constructed nine PPI networks displaying protein-protein interactions among the related genes. The nodes represent proteins, and the edges represent the interaction strength between two proteins. The proteins PFKFB3 (a), PDGFD (b), CXCR2 (c), CCL21 (d), SEMA3A (e), RYR3 (f), MEF2C (g), NEGR1(h) and TBC1D1 (i) which are located at the hub of the interaction network, are responsible for diaphragm fibrosis and atrophy.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Construct, Protein-Protein interactions

Journal: Scientific reports

Article Title: Single-nucleus transcriptomic profiling of the diaphragm during mechanical ventilation.

doi: 10.1038/s41598-024-82530-4

Figure Lengend Snippet: Fig. 8. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. qRT-PCR and Western blotting showing the expression of selected genes and corresponding proteins in the mechanically ventilated diaphragm and control ones. The bar graphs show the quantification of the mRNA expression of Pfkfb3 (a), Pdgfd (b), Cxcr2 (c), Negr1 (d), Sema3a (e), and Mef2c (f) normalized to that of GAPDH. Asterisks indicate significant differences (n = 3 in each group) (*P < 0.05). Western blotting analysis of the protein expression of PFKFB3 (g), PDGFD (h), CXCR2 (i), NEGR1 (j), SEMA3A (k), and MEF2C (l) normalized to that of β-actin as a loading control.

Article Snippet: After the semidry blotting procedure (50 min, 90 V), the membrane was incubated for 1 h at room temperature (RT) in 5% BSA blocking solution, followed by overnight incubation on a shaker at 4 °C with primary antibodies against PFKFB3 (bs-3528R, Boster Biological Technology Co., Ltd., Wuhan, China), PDGFD (bs-24572R, Boster), CXCR2 (bs-1629R, Boster), NEGR1 (bs-11095R, Boster), SEMA3A (bs-10468R, Boster), MEF2C (bs-4130R, Boster) and β-actin (66009-1-Ig, Proteintech, Wuhan, China) as a loading control.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: Experimental & molecular medicine

Article Title: PIK3R3 regulates PPARα expression to stimulate fatty acid β-oxidation and decrease hepatosteatosis.

doi: 10.1038/emm.2017.243

Figure Lengend Snippet: Figure 1 PIK3R3 expression is regulated by nutritional status and is upregulated in HFD-induced fatty liver mice. (a) Representative morphology and H&E, Oil Red O, Acadm and Cpt1a stained liver sections from C57BL/6J mice fed a normal chow or high-fat diet (HFD) for 8 weeks (n = 6/group). (b) Serum ketone body (top panel) and hepatic TG (bottom panel) levels of mice fed a normal chow diet or HFD. (c) Quantitative PCR (top panel) analysis and western blot (bottom panel) analysis showing the expression level changes of Pik3r3 in mice livers during HFD feeding. (d) Quantitative PCR (left) analysis and western blot (right) analysis showing expression levels of hepatic Pik3r3 in C57BL/6J mice subjected to ad libitum feeding, 24 h fasting and 24 h fasted/24 h re-fed conditions (n = 2/group). Data are expressed as the means ± s.e.m. *Po0.05; **Po0.01.

Article Snippet: Western blot detection was performed using antibodies specific for PIK3R3, GAPDH (Santa Cruz Biotechnology, CA, USA), HNF4α (Cell Signaling Technology, MA, USA), ACADM, CPT1a (Proteintech Group, Chicago, IL, USA), PPARα or PPARγ (Boster, Wuhan, China), which were previously tested and studied using both human and mouse samples.

Techniques: Expressing, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Experimental & molecular medicine

Article Title: PIK3R3 regulates PPARα expression to stimulate fatty acid β-oxidation and decrease hepatosteatosis.

doi: 10.1038/emm.2017.243

Figure Lengend Snippet: Figure 2 Overexpression of PIK3R3 regulated lipid metabolism in vitro and alleviated the fatty liver phenotype in vivo. (a) The indicated protein expression levels were detected by western blot after hepatic HepG2 and LO2 cells were transfected with the PIK3R3 overexpression vector (PIK3R3) or control vector (Vector). (b) Quantitative PCR analysis and chemical colorimetric diagnostic analysis showing the levels of genes involved in fatty acid oxidation in mice livers. (c) Mice were fed HFD for 8 weeks (n = 6/group) and injected with Ad-control or Ad-Pik3r3 as described in the Materials and methods. Mice were killed 5 days after the last injection. Representative morphology and H&E, Oil Red O, Acadm and Cpt1a stained sections of mouse livers. (d, e) Serum ketone body levels (d) and hepatic TG levels (e). Data are expressed as the means ± s.e.m. *Po0.05.

Article Snippet: Western blot detection was performed using antibodies specific for PIK3R3, GAPDH (Santa Cruz Biotechnology, CA, USA), HNF4α (Cell Signaling Technology, MA, USA), ACADM, CPT1a (Proteintech Group, Chicago, IL, USA), PPARα or PPARγ (Boster, Wuhan, China), which were previously tested and studied using both human and mouse samples.

Techniques: Over Expression, In Vitro, In Vivo, Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Diagnostic Assay, Injection, Staining

Journal: Experimental & molecular medicine

Article Title: PIK3R3 regulates PPARα expression to stimulate fatty acid β-oxidation and decrease hepatosteatosis.

doi: 10.1038/emm.2017.243

Figure Lengend Snippet: Figure 3 Down-regulation of PIK3R3 impairs lipid homeostasis in vitro and in vivo. (a) PIK3R3 was knocked down in hepatic HepG2 and LO2 cells by siRNA transfection; the indicated protein expression levels were detected by western blot. (b) Quantitative PCR analysis and chemical colorimetric diagnostic analysis showing the levels of genes involved in fatty acid oxidation in mice livers. (c) Mice were fed HFD for 8 weeks (n = 6/group) and injected with si-control or si-Pik3r3 as described in the Materials and methods. Mice were killed 5 days after the last injection. Representative morphology and H&E, Oil Red O, Acadm and Cpt1a stained sections of mouse livers. (d, e) Serum ketone body levels (d) and hepatic TG levels (e). Data are expressed as the means ± s.e.m. *Po0.05.

Article Snippet: Western blot detection was performed using antibodies specific for PIK3R3, GAPDH (Santa Cruz Biotechnology, CA, USA), HNF4α (Cell Signaling Technology, MA, USA), ACADM, CPT1a (Proteintech Group, Chicago, IL, USA), PPARα or PPARγ (Boster, Wuhan, China), which were previously tested and studied using both human and mouse samples.

Techniques: In Vitro, In Vivo, Transfection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Diagnostic Assay, Injection, Control, Staining

Journal: Experimental & molecular medicine

Article Title: PIK3R3 regulates PPARα expression to stimulate fatty acid β-oxidation and decrease hepatosteatosis.

doi: 10.1038/emm.2017.243

Figure Lengend Snippet: Figure 5 PPARα mediates the effects of PIK3R3 on cellular and hepatic lipid metabolism. (a) PIK3R3 was overexpressed in LO2 cells with or without the knockdown of PPARA; western blot analysis showing the protein levels of PIK3R3, PPARA, ACADM and CPT1A. (b) PIK3R3 was knocked down in HepG2 cells with or without the overexpression of PPARA; western blot analysis showing the protein levels of PIK3R3, PPARA, ACADM and CPT1A. (c) Mice were fed HFD for 8 weeks (n = 6/group) and injected with Ad-control, Ad-Pik3r3 or Ad-Pik3r3+si-Pparα as described in the Materials and methods. Mice were killed for further analysis 5 days after the last injection. Representative H&E, Oil Red O, Pparα, Acadm and Cpt1a stained sections of mouse livers. (d) Pik3r3 and Pparα expression levels were detected by western blot. (e) Chemical colorimetric diagnostic analysis showing the changes in hepatic TG. (f) Chemical colorimetric diagnostic analysis showing changes in serum ketone bodies. Data are expressed as the means ± s.e.m. *Po0.05.

Article Snippet: Western blot detection was performed using antibodies specific for PIK3R3, GAPDH (Santa Cruz Biotechnology, CA, USA), HNF4α (Cell Signaling Technology, MA, USA), ACADM, CPT1a (Proteintech Group, Chicago, IL, USA), PPARα or PPARγ (Boster, Wuhan, China), which were previously tested and studied using both human and mouse samples.

Techniques: Knockdown, Western Blot, Over Expression, Injection, Control, Staining, Expressing, Diagnostic Assay