Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 1. Gene and protein expression of LKB1 in GC muscles of all experimental groups. (A-D) RT-PCR and western blot (WB) analyses of LKB1 expression in the following mice: (A) C57BL/10 mdx mice (n=10) versus C57BL/10 WT mice (n=11) (*P<0.036); (B) D2 mdx mice (n=12) versus D2 WT mice (n=9) (*P<0.0192); (C) C57BL/10 mdx exercised (EXER) mice (n=4) versus C57BL/10 mdx sedentary (SED) mice (n=4) (*P=0.0153); (D) C57BL/10 mdx mice treated with metformin (MET) (n=4) versus C57BL/10 mdx mice (n=4) (*P=0.015). Blots were loaded by an operator unaware of the experimental groupings of samples. In A, the most representative blot of the three western blot experiments performed is shown because of the large number of samples belonging to Group 1. Data are mean+s.e.m. Significant differences were assessed by unpaired Student’s t-test.

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Expressing, Muscles, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 2. Immunofluorescence imaging for LKB1. GC muscle sections of 6-month-old BL10 mdx and WT mice were stained with antibodies against laminin as a sarcolemmal marker (green) and LKB1 (red). Nuclei were stained with DAPI (blue). RGB merged images of the three channels are shown at the bottom.

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Immunofluorescence, Imaging, Staining, Marker

Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 7. Gene expression and localization of LKB1 in WT and dystrophic murine muscle cells. (A) Lkb1 expression in 2B4 (WT) and SF1 (DMD) myoblasts (D0), and myocytes (D2-D11). Significant differences were assessed by unpaired Student’s t-test (*P≤0.05; ***P≤0.001). ns, not significant. (B) Immunofluorescence imaging for LKB1. D11 2B4 and SF1 cells were stained with antibodies against laminin as a sarcolemmal marker (green) and LKB1 (red). Nuclei were stained with DAPI (blue). RGB merged images of the three channels are shown on the right.

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Gene Expression, Expressing, Immunofluorescence, Imaging, Staining, Marker

Journal: Disease models & mechanisms

Article Title: LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model.

doi: 10.1242/dmm.049930

Figure Lengend Snippet: Fig. 8. Schematic summarizing the pathways involved in the LKB1- AMPK-HDAC axis in healthy and dystrophic muscle. In healthy muscle, LKB1 is activated in response to exercise, in turn stimulating the metabolic remodeling mediated by the key modulator AMPK. AMPK turns off the class II HDACs that are also inhibited via SIK phosphorylation by LKB1. In dystrophic muscle, this refined mechanism of regulation is altered owing to LKB1 impairment. This allows class II HDACs to carry on their epigenetic silencer action, thus inhibiting the expression of genes involved in oxidative metabolism (Mef2c).

Article Snippet: After three washes with PBS, sections and cells were blocked in saturation buffer (0.1%bovine serum albumin in PBS) and incubated with a rabbit polyclonal anti-laminin primary antibody (1:500; L9393S, Sigma-Aldrich) and mouse monoclonal anti-LKB1 primary antibody (1:50; sc-32245, Santa Cruz Biotechnology) for 2 h at RT in blocking buffer.

Techniques: Phospho-proteomics, Expressing

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a The UMAP plots of scRNA-seq analyses show the expression of Scgb1a1 , Foxj1 , or Stk11 in adult airway epithelial cells from adult lungs. b The pseudo-time trajectories show that the expression level of Scgb1a1 decreased during the ciliated cell differentiation process, whereas the expression levels of Foxj1 and Stk11 increased during the ciliated cell differentiation process. c The dot plot of Stk11 expression score in different cell types. The dot size represents the proportion of cells in a cluster that express the gene. The dot color corresponds to the average expression level of the gene. d E16.5 lungs were stained with antibodies against Acetylated-α-Tubulin and STK11. Scale bars: 25 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Expressing, Cell Differentiation, Staining

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a E16.5 lungs were stained with antibodies against FOXJ1 and SCGB1A1. b The proportion of FOXJ1 + cells (Left, n=3) and SCGB1A1 + cells (Right, n=4) in the intrapulmonary airways of E16.5 lungs. c Pregnant mice carrying Sox2 -CreER; Rosa26 -mTmG; Stk11 F/+ (Control) and Sox2 -CreER; Rosa26 -mTmG; Stk11 F/F ( Sox2 - Stk11 ) embryos were treated with tamoxifen at E13.5 and analyzed at E16.5. d E16.5 lungs were stained with antibodies against GFP, FOXJ1, and SCGB1A1. The yellow arrowheads indicate cells expressing both GFP and FOXJ1. e The proportion of FOXJ1 + GFP + cells to GFP + cells (Left) (Control, n=6; Sox2 - Stk11 , n=5) and SCGB1A1 + GFP + cells to GFP + cells (Right) (n=3) in the intrapulmonary airways of E16.5 lungs. f 10 weeks-old mice were treated with three doses of tamoxifen and analyzed after 30 days. g Lungs were stained with antibodies against GFP, FOXJ1, and SCGB1A1 at day 30. The yellow arrowheads indicate cells expressing both GFP and FOXJ1. h The proportion of FOXJ1 + GFP + cells to GFP + cells at day 30 (n=4). *, P <0.05; **, P <0.01; ***, P <0.001. Data shown in the graphs are means ± SEM. Student’s t -test. Scale bars: a , d : 25 μm, g : 20 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Staining, Control, Expressing

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a Heat map of the expression values of genes expressed differentially between E16.5 Shh - Stk11 lungs and control lungs. b Fold change of relative mRNA expression of airway epithelial cell genes in E16.5 Shh - Stk11 lungs compared to Control lungs (n=3). c Fold change of relative mRNA expression of cell-cycle genes in E16.5 Shh - Stk11 lungs compared to Control lungs (n=3). d Pregnant females were oral gavaged daily with a CDK4/6 inhibitor (PD0332991) each day from E12.5 to E14.5. Lungs were analyzed at E16.5. e Cell proliferation was analyzed by antibody staining against Ki67 and E-cadherin (E-cad). f The proportion of Ki67 + cells in the intrapulmonary airways of E16.5 lungs (vehicle Control, n=5; vehicle Shh - Stk11 , n=3; PD0332991 Control, n=7; PD0332991 Shh - Stk11 , n=8). g Immunofluorescence staining with antibodies against FOXJ1 and SCGB1A1 in lungs after either vehicle or PD0332991 treatment. h The proportion of FOXJ1 + cells (vehicle Control, n=3; vehicle Shh - Stk11 , n=4; PD0332991 Control, n=3; PD0332991 Shh - Stk11 , n=5) and the proportion of SCGB1A1 + cells (n=3) in the intrapulmonary airways of E16.5 lungs after either vehicle or PD0332991 treatment. i 10 week-old mice were treated with three doses of tamoxifen. Mice were treated with PD0332991 or vehicle every other day via oral gavage and fed with water containing IdU for 30 days before collecting lungs for analysis. j Immunofluorescence staining with antibodies against IdU and GFP in lungs after either vehicle or PD0332991 treatment for 30 days. k The proportion of IdU + cells in the GFP labeled intrapulmonary airway epithelium (n=3). l Immunofluorescence staining with antibodies against FOXJ1, GFP, and SCGB1A1 in lungs after either vehicle or PD0332991 treatment for 30 days. The yellow arrows indicate cells expressing both GFP and FOXJ1. m The proportion of FOXJ1 + GFP + cells to GFP + cells in lungs after either vehicle or PD0332991 treatment for 30 days (n=3). *, P <0.05; **, P <0.01; ***, P <0.001. Data shown in the graphs are means ± SEM. Student’s t -test. Scale bars: e , g : 11 μm; j , l : 20 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Expressing, Control, Staining, Immunofluorescence, Labeling

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a A schematic illustration showing the air-liquid interphase trachea culture system of this study. Adenovirus (Ad)-GFP, STK11 WT , or STK11 KD particles were added into the medium at day 0. The tracheal rudiments were cultured with different adenovirus for 5 days. b - c The proportion of Ki67 + cells in the epithelium of cultured tracheal rudiments at day 5 (c) were quantified by Ki67 immunostaining (b) (Ad-GFP Control, n=5; Ad-GFP Shh - Stk11 , n=6; Ad-STK11 WT Control, n=4; Ad-STK11 WT Shh - Stk11 , n=5; Ad-STK11 KD Control, n=4; Ad-STK11 KD Shh - Stk11 , n=6). d - f Cultured tracheal rudiments were stained with antibodies against FOXJ1 and GFP at day 5. g The proportion of FOXJ1 + cells in the epithelium of cultured tracheal rudiments at day 5 (Ad-GFP Control, n=3; Ad-GFP Shh - Stk11 , n=3; Ad-STK11 WT Control, n=3; Ad-STK11 WT Shh - Stk11 , n=3; Ad-STK11 KD Control, n=5; Ad-STK11 KD Shh - Stk11 , n=3). ns, not significant; *, P <0.05; **, P <0.01; ***, P <0.001. Data shown in the graphs are means ± SEM. Student’s t -test. Scale bars: 11 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Cell Culture, Immunostaining, Control, Staining

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a Immunofluorescence staining with antibodies against p-MARK3 and Acetylated-α-Tubulin in E16.5 lungs. Yellow arrows indicate ciliated cells expressing both Acetylated-α-Tubulin and p-MARK3. b E13.5 tracheal rudiments were cultured with Ad-GFP, Ad-MARK3 WT , or Ad-MARK3 CA viruses for 5 days. c - d Cultured tracheal rudiments were stained with antibodies against FOXJ1 at day 5 (c). The proportion of FOXJ1 + ciliated cells were quantified (Ad-GFP Control, n=3; Ad-GFP Shh - Stk11 , n=3; Ad-MARK3 WT Control, n=3; Ad-MARK3 WT Shh - Stk11 , n=4; Ad-MARK3 CA Control, n=4; Ad-MARK3 CA Shh - Stk11 , n=3) (d). e E16.5 lungs were stained with antibodies against p-ERK1/2 and SOX2. f Pregnant female mice were oral gavaged with p-ERK1/2 inhibitor PD0325901 or vehicle at E13.5. Lungs were analyzed at E16.5. g - h Immunofluorescence staining with antibodies against FOXJ1 in the vehicle-treated or PD0325901 treated lungs (g). The proportion of FOXJ1 + cells (n=3) and the proportion of SCGB1A1 + cells (n=3) in the intrapulmonary airways of E16.5 lungs were quantified (h). i E13.5 tracheal rudiments were cultured with vehicle or PD0325901 for 5 days. j - k Cultured tracheal rudiments were stained with antibodies against FOXJ1 at day 5 (j). The proportion of FOXJ1 + ciliated cells were quantified (vehicle Control, n=4; vehicle Shh - Stk11 , n=3; PD0325901 Control, n=3; PD0325901 Shh - Stk11 , n=5) (k). l A schematic illustration showing that STK11-mediated signaling cascade accounts for the normal program of ciliated cell differentiation in airways. ns, not significant; *, P <0.05; **, P <0.01; ***, P <0.001. Data shown in the graphs are means ± SEM. Student’s t -test. Scale bars: 11 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Immunofluorescence, Staining, Expressing, Cell Culture, Control, Cell Differentiation

Journal: bioRxiv

Article Title: STK11 is required for the normal program of ciliated cell differentiation in airways

doi: 10.1101/652107

Figure Lengend Snippet: a Immunofluorescence staining with antibodies against FOXJ1 and SCGB1A1 in the E16.5 lungs. White arrowheads indicate cells that do not express SCGB1A1 and FOXJ1. b The proportion of SCGB1A1 - FOXJ1 - cells (Left), and the proportion of FOXJ1 + cells (Right) in the intrapulmonary airways of E16.5 lungs (n=3). c Immunofluorescence staining with antibodies against FOXJ1 and SCGB1A1 in the E18.5 lungs. White arrowheads indicate cells that do not express SCGB1A1 and FOXJ1. d The proportion of SCGB1A1 - FOXJ1 - cells (Left), and the proportion of FOXJ1 + cells (Right) in the intrapulmonary airways of E18.5 lungs (n=3). e Immunofluorescence staining with antibodies against Acetylated-α-tubulin and JAG1 (JAGGED1) in E16.5 lungs. Yellow arrowheads indicate the Acetylated-α-tubulin - cells that express JAG1. f The proportion of JAG1 + cells in the intrapulmonary airways of E16.5 lungs (Control, n=4; Shh - Stk11 , n=4). g Immunofluorescence staining with antibodies against Acetylated-α-tubulin and JAG1 in E18.5 lungs. Yellow arrowheads indicate the Acetylated-α-tubulin - cells that express JAG1. h The proportion of JAG1 + cells in the intrapulmonary airways of E18.5 lungs (Control, n=4; Shh - Stk11 , n=5). ns, not significant; *, P <0.05; **, P <0.01; ***, P <0.001. Data shown in the graphs are means ± SEM. Student’s t -test. Scale bars: 11 μm.

Article Snippet: Immunofluorescence staining was performed using the following primary antibodies: rabbit anti-STK11 antibody (Cell Signaling, 13031, 1:500), mouse anti-Acetylated-α-Tubulin (Sigma, T6793, 1:2000), mouse anti-FOXJ1 (eBioscience, 14-9965, 1:500), mouse anti-MYB (Santa Cruz, sc-74512, 1:500), rabbit anti-Ki67 (Abcam, ab15580, 1:300), chicken anti-GFP antibody (Abcam, ab13970, 1:1000), rabbit anti-AMPKα (PRKAA1) antibody (Cell Signaling, 2535s, 1:500), rat anti-E-Cadherin (Invitrogen clone ECCD-2, 1:500), rabbit anti-PH3 (Millipore, 06-570, 1:100), rabbit anti-SCGB1A1 (Millipore, 07-623, 1:300), rabbit anti-phosphorylated MARKs antibody (Cell Signaling, 4836, 1:200), rabbit anti-Caspase3 antibody (Cell Signaling, 9664s, 1:300), goat anti-SOX2 antibody (Santa Cruz, sc-17320, 1:100), mouse anti-P63 (Santa Cruz, sc-8431, 1:500), rabbit anti-CGRP antibody (Sigma, C8198, 1:300), mouse anti-Flag antibody (Sigma, A2220, 1:200), rabbit anti-phosphorylated ERK1/2 antibody (Cell Signaling, 4370S, 1:200), rabbit anti-p-pRB antibody (Cell Signaling, 8516, 1:1600), rabbit anti-JAG1 antibody (Cell Signaling, 70109, 1:1000), and mouse anti-IdU antibody (BD Biosciences, 347580, 1:500).

Techniques: Immunofluorescence, Staining, Control

Journal: Molecular Medicine Reports

Article Title: Isoflurane suppresses the self-renewal of normal mouse neural stem cells in a p53-dependent manner by activating the Lkb1-p53-p21 signalling pathway

doi: 10.3892/mmr.2015.4387

Figure Lengend Snippet: Reverse-transcription quantitative polymerase chain reaction primers used in the present study.

Article Snippet: Rabbit anti-mouse LKB1 , (sc-5638) , Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) , WB (1:1,000).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Isoflurane suppresses the self-renewal of normal mouse neural stem cells in a p53-dependent manner by activating the Lkb1-p53-p21 signalling pathway

doi: 10.3892/mmr.2015.4387

Figure Lengend Snippet: List of primary antibodies used in the present study.

Article Snippet: Rabbit anti-mouse LKB1 , (sc-5638) , Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) , WB (1:1,000).

Techniques:

Journal: Molecular Medicine Reports

Article Title: Isoflurane suppresses the self-renewal of normal mouse neural stem cells in a p53-dependent manner by activating the Lkb1-p53-p21 signalling pathway

doi: 10.3892/mmr.2015.4387

Figure Lengend Snippet: Isoflurane activates Lkb1-p21 signalling in WT NSCs, although not in NE-4C (p53 −/− ) cells. (A) RT-qPCR analysis reveals that the mRNA expression levels of Lkb1 , p53 and p21 in WT NSCs treated with an IC 50 concentration of isoflurane were significantly higher compared with that in untreated cells ( ** P<0.01; # P>0.05 compared with the non-treated group; n=3). (B) RT-qPCR analysis indicates that the mRNA expression levels of p53 and p21 in isoflurane-treated NE-4C (p53 −/− ) cells were unchanged compared with the untreated cells ( ** P<0.01; * P<0.05; # P>0.05 compared with the non-treated group; n=3). (C) Western blotting indicates that the protein expression levels of Lkb1 and p21 in isoflurane-treated WT NSCs were significantly increased compared with untreated cells ( ** P<0.01; * P<0.05; # P>0.05 compared with the non-treated group; n=3). (D) Western blotting indicates that only the protein expression level of Lkb1 was increased in isoflurane-treated NE-4C (p53 −/− ) cells ( ** P<0.01; # P>0.05 compared with the non-treated group; n=3). GAPDH was used as a loading control. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT NSCs, wild-type neural stem cells.

Article Snippet: Rabbit anti-mouse LKB1 , (sc-5638) , Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) , WB (1:1,000).

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

Journal: Molecular Medicine Reports

Article Title: Isoflurane suppresses the self-renewal of normal mouse neural stem cells in a p53-dependent manner by activating the Lkb1-p53-p21 signalling pathway

doi: 10.3892/mmr.2015.4387

Figure Lengend Snippet: Immunofluorescence analysis of the Lkb1-p21 signalling pathway in WT NSCs. The protein expression levels of LKB1, p53-Ser-pho and p21 in isoflurane-treated WT NSCs were significantly increased compared with the untreated cells (scale bar, 50 µ m). FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; WT NSCs, wild-type neural stem cells; p53-Ser-pho, Ser15-phosphorylated p53.

Article Snippet: Rabbit anti-mouse LKB1 , (sc-5638) , Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) , WB (1:1,000).

Techniques: Immunofluorescence, Expressing

Journal: Molecular Medicine Reports

Article Title: Isoflurane suppresses the self-renewal of normal mouse neural stem cells in a p53-dependent manner by activating the Lkb1-p53-p21 signalling pathway

doi: 10.3892/mmr.2015.4387

Figure Lengend Snippet: Immunofluorescence analysis of the Lkb1-p21 signalling pathway in NE-4C (p53 −/− ) cells. Only Lkb1 expression was increased in isoflurane-treated NE-4C (p53 −/− ) cells compared with the untreated controls (scale bar, 50 µ m). FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole; WT NSCs, wild-type neural stem cells; p53-Ser-pho, Ser15-phosphorylated p53.

Article Snippet: Rabbit anti-mouse LKB1 , (sc-5638) , Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) , WB (1:1,000).

Techniques: Immunofluorescence, Expressing

Journal: PLoS ONE

Article Title: Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats

doi: 10.1371/journal.pone.0153169

Figure Lengend Snippet: Sequences of primers used for the quantitative real-time PCR.

Article Snippet: The primary antibodies were used at the following dilutions, and obtained from the indicated sources: anti-p-AMPKα (Thr172) diluted 1:3000 (Cell Signaling Technology, Beverly, USA), anti-AMPKα diluted 1:3000 (Cell Signaling Technology), anti-p-LKB1 (Ser428) diluted 1:3000 (Cell Signaling Technology), anti-LKB1 diluted 1:3000 (Cell Signaling Technology), and anti-β-actin diluted 1:5000 (Zhongshan Goldenbridge Biotech, Beijing, China).

Techniques:

Journal: PLoS ONE

Article Title: Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats

doi: 10.1371/journal.pone.0153169

Figure Lengend Snippet: (A) Cerebral cortex, (B) Hippocampus, (C) Thalamus, (D) Cerebellum and (E) Brainstem. Rats received saline (0.5 mL) or xylazine (5.2 mg/kg) intraperitoneally and then were sacrificed 10, 10, 20, 40 or 60 min later for control, Xyl1, Xyl2, Xyl3 or Xyl4, respectively. Total RNA was isolated and subjected to real-time PCR analysis. Each value of the expression levels of LKB1 was normalized to the expression levels of β-actin, and the control value was set to one. Data are presented as the means ± SEM, n = 6. * P < 0.05, ** P < 0.01 vs control.

Article Snippet: The primary antibodies were used at the following dilutions, and obtained from the indicated sources: anti-p-AMPKα (Thr172) diluted 1:3000 (Cell Signaling Technology, Beverly, USA), anti-AMPKα diluted 1:3000 (Cell Signaling Technology), anti-p-LKB1 (Ser428) diluted 1:3000 (Cell Signaling Technology), anti-LKB1 diluted 1:3000 (Cell Signaling Technology), and anti-β-actin diluted 1:5000 (Zhongshan Goldenbridge Biotech, Beijing, China).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: Xylazine Activates Adenosine Monophosphate-Activated Protein Kinase Pathway in the Central Nervous System of Rats

doi: 10.1371/journal.pone.0153169

Figure Lengend Snippet: (A) Cerebral cortex, (B) Hippocampus, (C) Thalamus, (D) Cerebellum and (E) Brainstem. Rats received saline (0.5 mL) or xylazine (5.2 mg/kg) intraperitoneally and then were sacrificed 10, 10, 20, 40 or 60 min later for control, Xyl1, Xyl2, Xyl3 or Xyl4, respectively. Western blot analyses were performed with anti-LKB1 and anti-phospho-LKB1 (Ser428). Data for densitometry represent the mean ± SEM obtained from six independent series of Western blotting for each animal group and time point after the procedure. * P < 0.05, ** P < 0.01 vs control. Representative blots are shown below graph.

Article Snippet: The primary antibodies were used at the following dilutions, and obtained from the indicated sources: anti-p-AMPKα (Thr172) diluted 1:3000 (Cell Signaling Technology, Beverly, USA), anti-AMPKα diluted 1:3000 (Cell Signaling Technology), anti-p-LKB1 (Ser428) diluted 1:3000 (Cell Signaling Technology), anti-LKB1 diluted 1:3000 (Cell Signaling Technology), and anti-β-actin diluted 1:5000 (Zhongshan Goldenbridge Biotech, Beijing, China).

Techniques: Western Blot