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: Oncotarget

Article Title: Establishment of mitochondrial pyruvate carrier 1 (MPC1) gene knockout mice with preliminary gene function analyses

doi: 10.18632/oncotarget.13210

Figure Lengend Snippet: A. Western blotting shows variable expressions of MPC1 and MPC2 in different organs. B. Corresponding quantified densitometry of the Western blotting results as shown in (A). Date are presented as mean ± SEM (n=3 separate animals). C. Relative MPC mRNA levels in the indicated mouse tissues. Values are presented as mean ± SEM (n=3 separate animals). Br, brain; Lu, lung; H, heart; Liv, liver; Kid, kidney.

Article Snippet: 5% fat-free milk was used to block the membrane for two hours at room temperature, followed by incubated overnight at 4°C with a rabbit anti-mouse BRP44L (1:500, Novus) and BRP44 antibody (1:500, abcam).

Techniques: Western Blot

Journal: Oncotarget

Article Title: Establishment of mitochondrial pyruvate carrier 1 (MPC1) gene knockout mice with preliminary gene function analyses

doi: 10.18632/oncotarget.13210

Figure Lengend Snippet: A. The mutagenesis of the target sites is revealed by the presence of a full-length PCR product with digestion products of 278 and 150 bps (Marker shows in the picture from top to bottom: 600bp, 500bp, 400bp, 300bp, 200bp, 100bp). B. Sequence data of mutations in several mutation mice. Red lines indicate deletion of target(s). The red arrows indicate the beginning sites of deletion. C. The deleted base sequences of each heterozygous model relative to the WT one. D. shows single strand DNA sequences of WT, NO.1 and NO17 heterozygous mutation mice. E. shows results of IHC, on which the WT mice show strong expression of MPC1 and MPC2 proteins, while the NO1 and NO17 mice show reduced levels of the expression of MPC1 and MPC2 proteins, and the MPC1−/− mouse shows very weak MPC1 protein expression, but relatively weak MPC2 protein expression. Values are presented as mean ± SEM (n=3 separate animals).

Article Snippet: 5% fat-free milk was used to block the membrane for two hours at room temperature, followed by incubated overnight at 4°C with a rabbit anti-mouse BRP44L (1:500, Novus) and BRP44 antibody (1:500, abcam).

Techniques: Mutagenesis, Marker, Sequencing, Expressing

Journal: Oncotarget

Article Title: Establishment of mitochondrial pyruvate carrier 1 (MPC1) gene knockout mice with preliminary gene function analyses

doi: 10.18632/oncotarget.13210

Figure Lengend Snippet: A. Mutations at loci chr12:+78920516 and chr12:+78920523. Three of the seven F0 MPC1-g1 mice showed a single base deletion, and 6 of the 10 MPC1-g2 mice revealed variable mutations, 5 mice with a single base deletion and another with multiple mutations. B. Mutations at the loci at chr12:-112097058 and chr12:-112097051. There was no any mutation discovered for the MPC1-g1. However, 4 of the 10 MPC1-g2 mice showed variable mutations. C. Mutations at the locus at chrX:+140020779. There was no any mutation for both the MPC1-g1 and MPC1-g2.

Article Snippet: 5% fat-free milk was used to block the membrane for two hours at room temperature, followed by incubated overnight at 4°C with a rabbit anti-mouse BRP44L (1:500, Novus) and BRP44 antibody (1:500, abcam).

Techniques: Mutagenesis

Journal: Oncotarget

Article Title: Establishment of mitochondrial pyruvate carrier 1 (MPC1) gene knockout mice with preliminary gene function analyses

doi: 10.18632/oncotarget.13210

Figure Lengend Snippet: A. qRT-PCR detection of gluconeogenesis genes expression in WT and the heterozygous MPC1 gene KO mice. B. Relative lipogenesis genes expression in WT and the heterozygous MPC1 gene KO mice. Values are presented as mean ± SEM (n=3 separate animals, * <0.05, ** <0.01, *** <0.001).

Article Snippet: 5% fat-free milk was used to block the membrane for two hours at room temperature, followed by incubated overnight at 4°C with a rabbit anti-mouse BRP44L (1:500, Novus) and BRP44 antibody (1:500, abcam).

Techniques: Quantitative RT-PCR, Expressing

Journal: Oncotarget

Article Title: Establishment of mitochondrial pyruvate carrier 1 (MPC1) gene knockout mice with preliminary gene function analyses

doi: 10.18632/oncotarget.13210

Figure Lengend Snippet: A. Glucose tolerance test (GTT) in MPC1 KO and WT mice fed in normal food (n = 3). B. Insulin tolerance test (ITT) in MPC1 KO and WT mice fed in normal food (n = 3, * <0.05, ** <0.01).

Article Snippet: 5% fat-free milk was used to block the membrane for two hours at room temperature, followed by incubated overnight at 4°C with a rabbit anti-mouse BRP44L (1:500, Novus) and BRP44 antibody (1:500, abcam).

Techniques:

Journal: Oncotarget

Article Title: Establishment of mitochondrial pyruvate carrier 1 (MPC1) gene knockout mice with preliminary gene function analyses

doi: 10.18632/oncotarget.13210

Figure Lengend Snippet: A. Mouse MPC1 loci and the locations of the two protospacer within the first exon of the MPC1 genes. The 20bp protospacers on the sense strand for the MPC1-g1 and MPC1-g2 are indicated by black lines and the adjacent PAM sequences are indicated by the red lines. B. RNA-guided gene targeting in mouse embryo includes the Cas9 and gRNA, which are two important parts and formed an integrated circle with correspondence promoters. The gRNA is specific recognizing the target and the Cas9 cleave the DNA chain. C. Histogram shows high efficiencies of CRISPR-mediated nuclease activity at the target loci.

Article Snippet: 5% fat-free milk was used to block the membrane for two hours at room temperature, followed by incubated overnight at 4°C with a rabbit anti-mouse BRP44L (1:500, Novus) and BRP44 antibody (1:500, abcam).

Techniques: CRISPR, Activity Assay