Journal: Nature Communications

Article Title: mTORC1 signaling facilitates differential stem cell differentiation to shape the developing murine lung and is associated with mitochondrial capacity

doi: 10.1038/s41467-022-34763-y

Figure Lengend Snippet: a Heatmap of gene expression of the glycolytic and related pathways from control and Rptor f/f ; Shh Cre/+ lungs at 11.5 days post coitus ( dpc ). The genes with significantly altered expression in the absence of Rptor were: Ldha (1), Aldoa (2), Pgk1 (3), Pfkl ( Pfkm ) (4), Tpi1 (5), Hk2 (6) and Pklr (7). The expression of these seven genes was decreased in Rptor -deficient lungs. b , c Gene Ontology (GO) pathway analysis of bulk RNA-Seq of control and Rptor f/f ; Shh Cre/+ lungs at 11.5 dpc . d Quantification of the relative ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) in purified lung epithelial cells derived from control and Rptor f/f ; Shh Cre/+ mice at 13.5 dpc ( n = 4 for each group). 16S rRNA and mtND1 represent the mitochondrial genes while Hk2 (hexokinase 2) represents the nuclear genes. e Quantification of the relative ratio of mtDNA to nDNA in purified lung epithelial cells derived from control and Rptor f/f ; Shh Cre/+ mice at 17.5 dpc ( n = 4 for each group). f Measurement of relative ATP production in purified lung epithelial cells derived from control and Rptor f/f ; Shh Cre/+ mice at 13.5 ( n = 4 for each group) and 17.5 ( n = 4 for each group) dpc . g Immunostaining of lung sections collected from Rptor f/f ; Shh Cre/+ mice and control littermates at 13.5 dpc . E-Cadherin (E-Cad) labeled all epithelial cells. The MTCO1 and MPC1 signals indicate the mitochondrial mass within individual epithelial cells. A reduction of MTCO1 and MPC1 signal in the lung epithelium of Rptor f/f ; Shh Cre/+ mice was detected. Scale bar = 5 μm. h Quantification of the relative MTCO1 and MPC1 signals in the lung epithelium ( n = 3 for each group). Five views per lung (a total of 15 views) were used for counting. The signal density in control and mutant lungs was measured using ImageJ, and then divided by the corresponding number of cells. i Immunostaining of lung sections collected from Rptor f/f ; Shh Cre/+ mice and control littermates at 17.5 dpc . MTCO1 signal was decreased in the lung epithelium of Rptor f/f ; Shh Cre/+ mice. Scale bar = 5 μm. j Quantification of the relative MTCO1 and MPC1 signals in the lung epithelium ( n = 3 for each group). Five views per lung (a total of 15 views) were used for counting as in ( h ). All values are mean ± SEM. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001; ns not significant (two-tailed, unpaired Student’s t -test). Source data are provided as a file.

Article Snippet: For immunofluorescence, the primary antibodies used were: chicken anti-GFP (1:200, abcam, Cat# ab13970; RRID:AB_300798), rabbit anti-NKX2.1 (1:100, Epitomics, Cat# 2044–1; RRID:AB_1267367), goat-anti-CC10 (1:200, Santa Cruz Biotechnology, Cat# sc-9773; RRID:AB_2183391), mouse anti-acetylated tubulin (1:200, MilliporeSigma, Cat# T6793; RRID:AB_477585), rabbit anti-prosurfactant protein C (proSP-C) (1:200, MilliporeSigma, Cat# AB3786; RRID:AB_91588), hamster anti-T1α (1:200, Developmental Studies Hybridoma Bank, Cat# 8.1.1; RRID:AB_531893), mouse anti-HOPX (1:100, Santa Cruz Biotechnology, Cat# sc-398703; RRID:AB_2687966), mouse anti-p63 (1:100, Santa Cruz Biotechnology, Cat# sc-8431; RRID:AB_628091), rabbit anti-MPC1 (1:100, MilliporeSigma, Cat# HPA045119; RRID:AB_10960421), rat anti-E cadherin (1:200, Life Technologies, Cat# 13-1900; RRID:AB_2533005), mouse anti-β-catenin (1:100, BD Transduction Laboratories, Cat# 610154; RRID:AB_397555), mouse anti-MTCO1 (1:100, abcam, Cat# ab14705; RRID:AB_2084810), mouse anti-GM130 (1:100, BD Biosciences, Cat# 610822; RRID:AB_398141), mouse anti-ACTA2 (1:200, Thermo Scientific Lab Vision, Cat# MS-113-P0; RRID:AB_64001), rat anti-PECAM-1 (CD31) (1:150, Santa Cruz Biotechnology, Cat# sc-18916; RRID:AB_627028), rabbit anti-PDGFRA (1:150, Cell Signaling Technology, Cat# 3164; RRID:AB_2162351), mouse anti-S6 Ribosomal Protein (1:100, Cell Signaling Technology, Cat# 2317; RRID:AB_2238583), rabbit anti-Phospho-S6 Ribosomal Protein (Ser235/236) (1:100, Cell Signaling Technology, Cat# 4856; RRID:AB_2181037), rabbit anti-SOX2 (D9B8N) (1:200, Cell Signaling Technology, Cat# 23064; RRID:AB_2714146), goat anti-SOX9 (1:200, R&D Systems, Cat# AF3075; RRID:AB_2194160), rabbit anti-pMLC (S19) (1:100, Cell Signaling Technology, Cat# 3671; RRID:AB_330248), rabbit anti-PKC Zeta (C-20) (1:100, Santa Cruz Biotechnology, Cat# sc-216; RRID:AB_2300359).

Techniques: Expressing, RNA Sequencing Assay, Purification, Derivative Assay, Immunostaining, Labeling, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: mTORC1 signaling facilitates differential stem cell differentiation to shape the developing murine lung and is associated with mitochondrial capacity

doi: 10.1038/s41467-022-34763-y

Figure Lengend Snippet: a , b Immunostaining of lung sections collected from control, Tfam f/f ; Shh Cre/+ and Cox10 f/f ; Shh Cre/+ mice at 13.5 days post coitus ( dpc ). E-Cadherin (E-Cad) labeled all epithelial cells. The MTCO1 and MPC1 signals indicate the mitochondrial mass within individual epithelial cells. A reduction in MTCO1 but not MPC1 signal in the lung epithelium of Tfam f/f ; Shh Cre/+ and Cox10 f/f ; Shh Cre/+ mice was observed. Scale bar = 10 μm. c , d Quantification of the relative MTCO1 and MPC1 signals in the lung epithelium ( n = 3 for each group). Five views per lung (a total of 15 views) were used for counting. The signal density in control and mutant lungs was measured using ImageJ, and then divided by the corresponding number of cells. e , f Quantification of the relative ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) in purified lung epithelial cells derived from control, Tfam f/f ; Shh Cre/+ ( n = 4 for each group) and Cox10 f/f ; Shh Cre/+ ( n = 3 for each group) mice at 13.5 dpc . 16S rRNA and mtND1 represent the mitochondrial genes while Hk2 (hexokinase 2) represents the nuclear genes. g Measurement of relative ATP production in purified lung epithelial cells derived from control, Tfam f/f ; Shh Cre/+ ( n = 3 for each group) and Cox10 f/f ; Shh Cre/+ ( n = 3 for each group) mice at 13.5 dpc . h Whole-lung imaging of dissected lungs from Shh Cre/+ ; ROSA26 mTmG/ + (control), Tfam f/f ; Shh Cre/+ ; ROSA26 mTmG/ + and Cox10 f/f ; Shh Cre/+ ; ROSA26 mTmG/ + mice at 12.5–14.5 dpc . GFP was activated from the ROSA26 mTmG allele in all lung epithelial cells by Shh Cre ; tdTomato marked all non-epithelial cells. Epithelial branching was perturbed in Tfam- and Cox10 -deficient lungs. Scale bar = 1 mm. i Whole-mount immunostaining of dissected lungs from Shh Cre/+ ; ROSA26 mTmG/ + (control), Tfam f/f ; Shh Cre/+ ; ROSA26 mTmG/ + and Cox10 f/f ; Shh Cre/+ ; ROSA26 mTmG/ + mice at 12.75, 13.5 and 15.5 dpc . SOX9 labeled the distal airway epithelium, while SOX2 marked the proximal airway epithelium. Perturbation of SOX9 to SOX2 differentiation could be detected as early as 12.75 dpc in Cox10 f/f ; Shh Cre/+ ; ROSA26 mTmG/ + mice. Scale bar = 1 mm. All values are mean ± SEM. (**) p < 0.01; (***) p < 0.001; ns not significant (two-tailed, unpaired Student’s t -test). Source data are provided as a file.

Article Snippet: For immunofluorescence, the primary antibodies used were: chicken anti-GFP (1:200, abcam, Cat# ab13970; RRID:AB_300798), rabbit anti-NKX2.1 (1:100, Epitomics, Cat# 2044–1; RRID:AB_1267367), goat-anti-CC10 (1:200, Santa Cruz Biotechnology, Cat# sc-9773; RRID:AB_2183391), mouse anti-acetylated tubulin (1:200, MilliporeSigma, Cat# T6793; RRID:AB_477585), rabbit anti-prosurfactant protein C (proSP-C) (1:200, MilliporeSigma, Cat# AB3786; RRID:AB_91588), hamster anti-T1α (1:200, Developmental Studies Hybridoma Bank, Cat# 8.1.1; RRID:AB_531893), mouse anti-HOPX (1:100, Santa Cruz Biotechnology, Cat# sc-398703; RRID:AB_2687966), mouse anti-p63 (1:100, Santa Cruz Biotechnology, Cat# sc-8431; RRID:AB_628091), rabbit anti-MPC1 (1:100, MilliporeSigma, Cat# HPA045119; RRID:AB_10960421), rat anti-E cadherin (1:200, Life Technologies, Cat# 13-1900; RRID:AB_2533005), mouse anti-β-catenin (1:100, BD Transduction Laboratories, Cat# 610154; RRID:AB_397555), mouse anti-MTCO1 (1:100, abcam, Cat# ab14705; RRID:AB_2084810), mouse anti-GM130 (1:100, BD Biosciences, Cat# 610822; RRID:AB_398141), mouse anti-ACTA2 (1:200, Thermo Scientific Lab Vision, Cat# MS-113-P0; RRID:AB_64001), rat anti-PECAM-1 (CD31) (1:150, Santa Cruz Biotechnology, Cat# sc-18916; RRID:AB_627028), rabbit anti-PDGFRA (1:150, Cell Signaling Technology, Cat# 3164; RRID:AB_2162351), mouse anti-S6 Ribosomal Protein (1:100, Cell Signaling Technology, Cat# 2317; RRID:AB_2238583), rabbit anti-Phospho-S6 Ribosomal Protein (Ser235/236) (1:100, Cell Signaling Technology, Cat# 4856; RRID:AB_2181037), rabbit anti-SOX2 (D9B8N) (1:200, Cell Signaling Technology, Cat# 23064; RRID:AB_2714146), goat anti-SOX9 (1:200, R&D Systems, Cat# AF3075; RRID:AB_2194160), rabbit anti-pMLC (S19) (1:100, Cell Signaling Technology, Cat# 3671; RRID:AB_330248), rabbit anti-PKC Zeta (C-20) (1:100, Santa Cruz Biotechnology, Cat# sc-216; RRID:AB_2300359).

Techniques: Immunostaining, Labeling, Mutagenesis, Purification, Derivative Assay, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: mTORC1 signaling facilitates differential stem cell differentiation to shape the developing murine lung and is associated with mitochondrial capacity

doi: 10.1038/s41467-022-34763-y

Figure Lengend Snippet: a Immunostaining of lung sections collected from control, Tfam f/f ; Shh Cre/+ and Cox10 f/f ; Shh Cre/+ mice at 18.5 days post coitus ( dpc ). E-Cadherin (E-Cad) labeled all epithelial cells. The MTCO1 and MPC1 signals indicate the mitochondrial mass within individual epithelial cells. A reduction in MTCO1 but not MPC1 signal was detected in both Tfam f/f ; Shh Cre/+ and Cox10 f/f ; Shh Cre/+ lungs. Scale bar = 10 μm. b Immunostaining of lung sections collected from control, Tfam f/f ; Shh Cre/+ and Cox10 f/f ; Shh Cre/+ mice at 18.5 dpc . SPC labeled alveolar epithelial type II (AT2) cells, while T1α marked alveolar epithelial type I (AT1) cells. Scale bar = 25 μm. c Quantification of the relative MTCO1 and MPC1 signals in the lung epithelium ( n = 3 for each group) at 18.5 dpc . Five views per lung (a total of 15 views) were used for counting. The signal density in control and mutant lungs was measured using ImageJ, and then divided by the corresponding number of cells. d Quantification of the percentage of HOPX + (AT1) cells and SPC + (AT2) cells in the saccules ( n = 3 for each group). Five views per lung (a total of 15 views) were used for counting. e Quantification of the percentage of T1α + cells in the non-saccular epithelium (excluding the trachea and main stem bronchi) ( n = 3 for each group). Five views per lung (a total of 15 views) were used for counting. f Schematic diagram of how the mTORC1-mitochondria axis regulates lung branching morphogenesis. A pool of SOX9 + progenitors at the distal epithelium (tip progenitors) expands to produce SOX9 + transition progenitors, which generate SOX2 + progeny in the conducting airways. Subsequently, SOX9 + tip progenitors produce progeny for forming saccules. Production of the conducting airways and saccules is also closely associated with proper cell differentiation. Club cells (CC10 + ), ciliated cells (Ac-tub + ) and goblet cells line the conducting airways while saccules contain AT1 (HOPX + ; T1α + ) and AT2 cells (SPC + ) cells. SOX9 + progenitors differentiate into HOPX + cells that are adjacent to the SOX2 + conducting airways. SOX9 + progenitors are located distal to HOPX + cells and differentiate into SPC + cells within saccules. Disruption of mTORC1 signaling leads to reduced mitochondrial capacity. SOX9 + tip progenitors expand to generate SOX9 + transition progenitors. However, transition progenitors fail to produce SOX2 + progeny and thus the conducting airways are missing. Instead, lung cysts are generated. SOX9 + progenitors produce saccule-like structures on the surface or the edge of lung cysts. All values are mean ± SEM. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001; ns not significant (two-tailed, unpaired Student’s t -test). Source data are provided as a file.

Article Snippet: For immunofluorescence, the primary antibodies used were: chicken anti-GFP (1:200, abcam, Cat# ab13970; RRID:AB_300798), rabbit anti-NKX2.1 (1:100, Epitomics, Cat# 2044–1; RRID:AB_1267367), goat-anti-CC10 (1:200, Santa Cruz Biotechnology, Cat# sc-9773; RRID:AB_2183391), mouse anti-acetylated tubulin (1:200, MilliporeSigma, Cat# T6793; RRID:AB_477585), rabbit anti-prosurfactant protein C (proSP-C) (1:200, MilliporeSigma, Cat# AB3786; RRID:AB_91588), hamster anti-T1α (1:200, Developmental Studies Hybridoma Bank, Cat# 8.1.1; RRID:AB_531893), mouse anti-HOPX (1:100, Santa Cruz Biotechnology, Cat# sc-398703; RRID:AB_2687966), mouse anti-p63 (1:100, Santa Cruz Biotechnology, Cat# sc-8431; RRID:AB_628091), rabbit anti-MPC1 (1:100, MilliporeSigma, Cat# HPA045119; RRID:AB_10960421), rat anti-E cadherin (1:200, Life Technologies, Cat# 13-1900; RRID:AB_2533005), mouse anti-β-catenin (1:100, BD Transduction Laboratories, Cat# 610154; RRID:AB_397555), mouse anti-MTCO1 (1:100, abcam, Cat# ab14705; RRID:AB_2084810), mouse anti-GM130 (1:100, BD Biosciences, Cat# 610822; RRID:AB_398141), mouse anti-ACTA2 (1:200, Thermo Scientific Lab Vision, Cat# MS-113-P0; RRID:AB_64001), rat anti-PECAM-1 (CD31) (1:150, Santa Cruz Biotechnology, Cat# sc-18916; RRID:AB_627028), rabbit anti-PDGFRA (1:150, Cell Signaling Technology, Cat# 3164; RRID:AB_2162351), mouse anti-S6 Ribosomal Protein (1:100, Cell Signaling Technology, Cat# 2317; RRID:AB_2238583), rabbit anti-Phospho-S6 Ribosomal Protein (Ser235/236) (1:100, Cell Signaling Technology, Cat# 4856; RRID:AB_2181037), rabbit anti-SOX2 (D9B8N) (1:200, Cell Signaling Technology, Cat# 23064; RRID:AB_2714146), goat anti-SOX9 (1:200, R&D Systems, Cat# AF3075; RRID:AB_2194160), rabbit anti-pMLC (S19) (1:100, Cell Signaling Technology, Cat# 3671; RRID:AB_330248), rabbit anti-PKC Zeta (C-20) (1:100, Santa Cruz Biotechnology, Cat# sc-216; RRID:AB_2300359).

Techniques: Immunostaining, Labeling, Mutagenesis, Cell Differentiation, Generated, Two Tailed Test

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: