Journal: Scientific Reports

Article Title: Effects of glucose metabolism pathways on nuclear and cytoplasmic maturation of pig oocytes

doi: 10.1038/s41598-020-59709-6

Figure Lengend Snippet: Effects of down regulating related genes in DOs or CCM on in vitro maturation of pig oocytes. Graph ( A ) shows percentages of MII oocytes after DOs were cultured for 48 h in the NCSU-23 medium containing 0, 10 or 15 mM pyruvate (Pyr) with 10, 25 or 50 µM 4-CIN (CIN) or with 0.01, 0.03 or 0.05 µM rotenone (Rot). Graph B shows percentages of MII oocytes after DOs were cultured for 48 h in the NCSU-23 medium containing 0, 5 or 10 mM lactate (Lac) with 10, 25 or 50 mM sodium oxamate (Oxm) or with 10, 20 or 50 µM 4-CIN. Graphs ( C–E ) show MII percentages of cocultured DOs (Left Y axis) and levels of MPC1, NDUFV1 and LDHB (Right Y axis) after transfection of CCM with negative control (NC) siRNA, MPC1 siRNA (MP1, 2 or 3), NDUFV1 siRNA (ND1, 2 or 3), or LDHB siRNA (LD1, 2 or 3), respectively. Graphs ( F,G ) show MII percentages of DOs after microinjection with NC, MP3, ND1 or LD2 siRNAs. While DOs and the CCM transfected/injected with MPC1 or NDUFV1 siRNAs were cultured in NCSU-23 containing 15 mM pyruvate, DOs and the CCM transfected/injected with LDHB siRNA were cultured in NCSU-23 containing 10 mM lactate. In oocyte maturation experiments, each treatment was repeated 4 times with each replicate containing about 20 oocytes. Graphs ( H,I ) show ATP concentrations (ng/ml) in medium conditioned with CCM in NCSU-23 medium containing 5.6 mM glucose and 15 mM pyruvate, respectively, after transfection with G6PD siRNA-3 (G6-3), GAPDH siRNA-3 (GA-3) or MPC1 siRNA-3 (MP3). Each treatment was repeated 3 times with each replicate containing medium recovered from one culture well on different experimental days. a–f: Values with a different letter above bars differ significantly (P < 0.05).

Article Snippet: The primary antibodies used included rabbit anti-G6PD monoclonal antibodies (1:1000, ab993, Abcam Co., Ltd, Beijing, China), mouse anti-GAPDH monoclonal antibodies (1:1000, CW0100A, CWBio Co., Ltd, Beijing, China), rabbit anti-LDHB polyclonal antibodies (1:1000, 14824-1-AP, Proteintech Co., Ltd, Wuhan, China), rabbit anti-MPC1 polyclonal antibodies (1:200, TA315496S, ORIGENE Co., Ltd, Beijing, China), rabbit anti-NDUFV1 polyclonal antibodies (1:500, 11238-1-AP, Proteintech Co., Ltd, Wuhan, China), mouse anti-β-tubulin monoclonal antibodies (1:1000, 05–661, Merck Millipore), and mouse anti-β-actin monoclonal antibodies (1:1000, CW00096M, CWBio Co., Ltd).

Techniques: In Vitro, Cell Culture, Transfection, Negative Control, Injection

Journal: Scientific Reports

Article Title: Effects of glucose metabolism pathways on nuclear and cytoplasmic maturation of pig oocytes

doi: 10.1038/s41598-020-59709-6

Figure Lengend Snippet: Sequences of the sense strands of siRNAs targeting different genes.

Article Snippet: The primary antibodies used included rabbit anti-G6PD monoclonal antibodies (1:1000, ab993, Abcam Co., Ltd, Beijing, China), mouse anti-GAPDH monoclonal antibodies (1:1000, CW0100A, CWBio Co., Ltd, Beijing, China), rabbit anti-LDHB polyclonal antibodies (1:1000, 14824-1-AP, Proteintech Co., Ltd, Wuhan, China), rabbit anti-MPC1 polyclonal antibodies (1:200, TA315496S, ORIGENE Co., Ltd, Beijing, China), rabbit anti-NDUFV1 polyclonal antibodies (1:500, 11238-1-AP, Proteintech Co., Ltd, Wuhan, China), mouse anti-β-tubulin monoclonal antibodies (1:1000, 05–661, Merck Millipore), and mouse anti-β-actin monoclonal antibodies (1:1000, CW00096M, CWBio Co., Ltd).

Techniques:

Journal: eLife

Article Title: Acquisition of cellular properties during alveolar formation requires differential activity and distribution of mitochondria

doi: 10.7554/eLife.68598

Figure Lengend Snippet: ( A ) Immunostaining of lung sections collected from Sox9 Cre/+ ; ROSA26 mTmG/+ mice at 18.5 days post coitus ( dpc ) and different postnatal (P) stages as indicated. The GFP signal identified alveolar epithelial cells (alveolar type I [AT1] and alveolar type II [AT2] cells) while myofibroblasts were characterized by smooth muscle actin (SMA) expression. Moreover, mitochondria were labeled by MPC1; the trans-Golgi network was visualized by TGN38. Enhanced MPC1 signal was distributed nonuniformly in both alveolar epithelial cells and myofibroblasts. ( B ) Transmission electron micrographs of lungs collected from wild-type mice at P3. Prominent features in a given lung cell type include lamellar bodies in AT2 cells, elongated cell membrane in AT1 cells, and actin bundles and collagen fibers in myofibroblasts. RBC, red blood cell.

Article Snippet: Antibody , Anti-MPC1 (BRP44L) (rabbit polyclonal) , MilliporeSigma , Cat# HPA045119; RRID: AB_10960421 , IF (1:100).

Techniques: Immunostaining, Expressing, Labeling, Transmission Assay, Membrane

Journal: eLife

Article Title: Acquisition of cellular properties during alveolar formation requires differential activity and distribution of mitochondria

doi: 10.7554/eLife.68598

Figure Lengend Snippet: ( A ) Schematic diagram of the time course of postnatal (P) administration of tamoxifen and harvest of mouse lungs. ( B ) Immunostaining of lungs collected from CAGG CreER/+ ; ROSA26 mTmG/+ (control) and Tfam f/f ; CAGG CreER/+ ; ROSA26 mTmG/+ mice at P10 that had received tamoxifen at P0. The GFP signal represents sites of induced CreER activity. Nuclear NKX2.1 staining marked all lung epithelial cells while PDGFRA immunoreactivity labeled mesenchymal fibroblasts/myofibroblasts. ( C ) Hematoxylin and eosin-stained lung sections of control and Tfam f/f ; CAGG CreER/+ ; ROSA26 mTmG/+ mice at P10. Histological analysis revealed the presence of enlarged saccules and retarded development of secondary septa in the mutant lungs. ( D ) Measurement of the mean linear intercept (MLI) in control and Tfam f/f ; CAGG CreER/+ ; ROSA26 mTmG/+ lungs at P10 (n = 4 for each group). The MLI was increased in Tfam -deficient lungs. ( E ) Immunostaining of lung sections collected from control and Tfam f/f ; CAGG CreER/+ ; ROSA26 mTmG/+ mice at P10. Smooth muscle actin (SMA) expression was characteristic of myofibroblasts and phalloidin stained the actin filaments. ( F ) Quantification of the relative ratio of mitochondrial DNA (mtDNA), 16S rRNA, and mitochondrially encoded NADH dehydrogenase 1 (mtND1), to nDNA (nuclear DNA), hexokinase (Hk2), in lysates derived from control and Tfam f/f ; CAGG CreER/+ ; ROSA26 mTmG/+ lungs (n = 4 for each group). ( G ) Immunostaining of lung sections collected from control and Tfam f/f ; CAGG CreER/+ ; ROSA26 mTmG/+ mice at P10. MPC1 antibodies marked mitochondria; MTCO1 antibodies detected cytochrome c oxidase, the expression of which was controlled by Tfam . All values are mean ± SEM. *p<0.05; **p<0.01 (unpaired Student’s t -test). Figure 2—source data 1. Mean linear intercept and relative mitochondrial DNA (mtDNA)/nuclear DNA (nDNA) ratio.

Article Snippet: Antibody , Anti-MPC1 (BRP44L) (rabbit polyclonal) , MilliporeSigma , Cat# HPA045119; RRID: AB_10960421 , IF (1:100).

Techniques: Immunostaining, Control, Activity Assay, Staining, Labeling, Mutagenesis, Expressing, Derivative Assay

Journal: eLife

Article Title: Acquisition of cellular properties during alveolar formation requires differential activity and distribution of mitochondria

doi: 10.7554/eLife.68598

Figure Lengend Snippet: ( A ) Immunostaining of lung sections collected from Sox9 Cre/+ ; ROSA26 mTmG/+ (control) and Tfam f/f ; Sox9 Cre/+ ; ROSA26 mTmG/+ mice at 16.5 days post coitus ( dpc ). MPC1 antibodies marked mitochondria while MTCO1 antibodies detected cytochrome c oxidase, the expression of which was controlled by Tfam . ( B ) Immunostaining of fibroblasts derived from control or Tfam f/f ; Pdgfra Cre/+ lungs at postnatal (P) day 3. ( C ) Immunostaining of fibroblasts derived from control or Rhot1 f/f ; Pdgfra Cre/+ lungs at P5.

Article Snippet: Antibody , Anti-MPC1 (BRP44L) (rabbit polyclonal) , MilliporeSigma , Cat# HPA045119; RRID: AB_10960421 , IF (1:100).

Techniques: Immunostaining, Control, Expressing, Derivative Assay

Journal: eLife

Article Title: Acquisition of cellular properties during alveolar formation requires differential activity and distribution of mitochondria

doi: 10.7554/eLife.68598

Figure Lengend Snippet: ( A ) Schematic diagram of the time course of postnatal (P) administration of tamoxifen and harvest of mouse lungs. ( B ) Hematoxylin and eosin-stained lung sections of control and Rptor f/f ; CAGG CreER/+ mice at P10. Histological analysis revealed the presence of enlarged saccules and retarded development of secondary septa in the mutant lungs. ( C ) Measurement of the mean linear intercept (MLI) in control and Rptor f/f ; CAGG CreER/+ lungs at P10 (n = 5 for each group). The MLI was increased in Rptor -deficient lungs. ( D ) Immunostaining of lung sections collected from control and Rptor f/f ; CAGG CreER/+ ; ROSA26 mTmG/+ mice at P10. Smooth muscle actin (SMA) expression was characteristic of myofibroblasts and phalloidin stained the actin filaments. ( E ). Quantification of the protein levels of RPS6 and phosphorylated RPS6 (p-RPS6) in lung lysates derived from control and Rptor f/f ; CAGG CreER/+ ; ROSA26 mTmG/+ lungs (n = 5 for each group). ( F ) Quantification of the relative ratio of mitochondrial DNA (mtDNA), 16S rRNA, and mitochondrially encoded NADH dehydrogenase 1 (mtND1), to nuclear DNA (nDNA), hexokinase 2 (Hk2), in lysates derived from control and Rptor f/f ; CAGG CreER/+ ; ROSA26 mTmG/+ lungs (n = 5 for each group). ( G ) Immunostaining of lung sections collected from control and Rptor f/f ; CAGG CreER/+ ; ROSA26 mTmG/+ mice at P10. MPC1 antibodies marked mitochondria; MTCO1 antibodies detected cytochrome c oxidase, the expression of which was controlled by Tfam . All values are mean ± SEM. p<0.05; **p<0.01; ***p<0.001 (unpaired Student’s t -test). Figure 7—source data 1. Mean linear intercept, relative protein levels, and relative mitochondrial DNA (mtDNA)/nuclear DNA (nDNA) ratio.

Article Snippet: Antibody , Anti-MPC1 (BRP44L) (rabbit polyclonal) , MilliporeSigma , Cat# HPA045119; RRID: AB_10960421 , IF (1:100).

Techniques: Staining, Control, Mutagenesis, Immunostaining, Expressing, Derivative Assay

Journal: eLife

Article Title: Acquisition of cellular properties during alveolar formation requires differential activity and distribution of mitochondria

doi: 10.7554/eLife.68598

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-MPC1 (BRP44L) (rabbit polyclonal) , MilliporeSigma , Cat# HPA045119; RRID: AB_10960421 , IF (1:100).

Techniques: Electron Microscopy, Protease Inhibitor, Imaging, cDNA Synthesis, Recombinant, Plasmid Preparation, Cloning, Modification, Transduction, Software