Journal: Scientific Reports

Article Title: Female mice lacking Pald1 exhibit endothelial cell apoptosis and emphysema

doi: 10.1038/s41598-017-14894-9

Figure Lengend Snippet: Increased apoptosis and proliferation of endothelial cells in Pald1 knock-out female lungs. ( a , b ) Quantification of cleaved caspase-3 positive (CC3+) cells in 4 and 19-week old lungs revealed a significant increase in the number of cleaved caspase-3 positive endothelial cells (Erg positive) at 4 weeks of age and at 19 weeks in female ( a , n = 3–4), but not in male Pald1 −/− mice ( b , n = 3–4). There was no significant increase in non-endothelial cells (Erg negative) at both 4 and 19 weeks. ( c , d ) Quantification of Ki67 positive cells in 4 and 19-week old lungs revealed an increased number of Ki67-positive endothelial cells (Erg positive) in female ( c , n = 3–4), but not in male Pald1 −/− mice ( d , n = 3–4). There was no significant increase in non-endothelial cells (Erg negative) at both 4 and 19 weeks. Error bars: SD, t-test between genotypes within each age group. * = p ≤ 0.05, **** = p ≤ 0.0001.

Article Snippet: After overnight incubation in 30% sucrose in PBS, lung sections of 5–10 µm were cut, blocked (3% BSA, 0.1% Triton x-100, 5% Normal Donkey serum [Jackson Immunoresearch], 5% Normal Mouse serum [Invitrogen] in PBS) and stained with rabbit anti-ERG (1:100, Abcam, ab92513), mouse anti-ERG (1:100, Abcam, ab140520), mouse anti-αSMA (1:100, Sigma, C6198), rabbit anti-prosurfactant Protein C (1:100, Abcam, ab40879), mouse anti-cytokeratin (1:100, Sigma, P2871), rat anti-CD68 (1:100, AbD serotec, MCA1957), rabbit anti-cleaved caspase 3 Ab-5 (1:3000, Neomarkers, RB-1611-P1), and mouse anti-ki67 (1:100, Dako, M7240) in combination with appropriate fluorophore-coupled secondary antibodies.

Techniques: Knock-Out

Journal: Head & Face Medicine

Article Title: Expression of caspase-3, p53 and Bcl-2 in generalized aggressive periodontitis

doi: 10.1186/1746-160X-2-17

Figure Lengend Snippet: Significant nuclear staining for p53 was observed in the mononuclear inflammatory cells in the GAP group specimens. (×200 p53 immunostaining).

Article Snippet: The sections were then incubated with primary antibodies for p53 (Ready-to-use Ab-5, NeoMarkers, Fremont, CA, USA), Bcl-2 (Ready-to-use Ab-1, NeoMarkers, Fremont, CA, USA), and caspase-3 (Ready-to-use Ab-5, NeoMarkers, Fremont, CA, USA) for 2 hours in a humidified chamber at room temperature.

Techniques: Staining, Immunostaining

Journal: Head & Face Medicine

Article Title: Expression of caspase-3, p53 and Bcl-2 in generalized aggressive periodontitis

doi: 10.1186/1746-160X-2-17

Figure Lengend Snippet: The distribution of stained cells in GAP patients and the controls.

Article Snippet: The sections were then incubated with primary antibodies for p53 (Ready-to-use Ab-5, NeoMarkers, Fremont, CA, USA), Bcl-2 (Ready-to-use Ab-1, NeoMarkers, Fremont, CA, USA), and caspase-3 (Ready-to-use Ab-5, NeoMarkers, Fremont, CA, USA) for 2 hours in a humidified chamber at room temperature.

Techniques: Staining

Journal:

Article Title: MUC1 oncoprotein promotes autophagy in a survival response to glucose deprivation

doi:

Figure Lengend Snippet: MUC1 blocks increases in ROS and necrosis of HCT116 cells in response to glucose deprivation. (A) HCT116/vector and HCT116/MUC1 cells were cultured in 25 or 1 mM glucose for 24 h. The cells were incubated with DCFH-DA for 20 min. Fluorescence of oxidized DCF was measured by flow cytometry. (B) The results are expressed as the relative ROS level (mean ± SD of three separate experiments) for cells in 1 mM glucose (open bars) compared to that in 25 mM glucose (solid bars). (C) HCT116/vector (clones A and B) and HCT116/MUC1 (clones A and B) cells were cultured in 25 or 1 mM glucose for 24 h. The cells were stained with PI and analyzed by flow cytometry. (D) The results are expressed as the percentage (mean ± SD of three separate experiments) necrotic cells for 25 mM (solid bars) or 1 mM (open bars) glucose.

Article Snippet: Cells were lysed as described ( 46 ) and analyzed by immunoblotting with anti-MUC1-C (Ab5; NeoMarkers Inc., Fremont, CA), anti-β-actin (Sigma), anti-MAP1LC3 (NanoTools; Freiburg, Germany), anti-phospho-Akt (anti-p-Akt) anti-Akt, anti-phospho-AMPKα (Thr-172) (p-AMPK) and anti-AMPKα (Cell Signaling Inc.).

Techniques: Plasmid Preparation, Cell Culture, Incubation, Fluorescence, Flow Cytometry, Clone Assay, Staining

Journal:

Article Title: MUC1 oncoprotein promotes autophagy in a survival response to glucose deprivation

doi:

Figure Lengend Snippet: MUC1 blocks ROS-induced depletion of ATP in the response to glucose deprivation. (A) HCT116/vector (open bars) and HCT116/MUC1 (solid bars) cells were cultured in 1 mM glucose for 0, 24, 48 and 72 h. Intracellular ATP levels are expressed as mean ± SD of three separate determinations relative to that at 0 h. (B–D) HCT116/vector cells were cultured in 25 mM glucose (solid bars), 1 mM glucose (open bars) or 1 mM glucose and 500 U/ml catalase (shaded bars) for 24 h. The cells were incubated with DCFH-DA and analyzed by flow cytometry (B). The results are expressed as the relative ROS level (mean ± SD of three separate experiments) compared to that obtained in 25 mM glucose (C). Intracellular ATP levels are expressed as mean ± SD of three experiments relative to that at 0 h (D).

Article Snippet: Cells were lysed as described ( 46 ) and analyzed by immunoblotting with anti-MUC1-C (Ab5; NeoMarkers Inc., Fremont, CA), anti-β-actin (Sigma), anti-MAP1LC3 (NanoTools; Freiburg, Germany), anti-phospho-Akt (anti-p-Akt) anti-Akt, anti-phospho-AMPKα (Thr-172) (p-AMPK) and anti-AMPKα (Cell Signaling Inc.).

Techniques: Plasmid Preparation, Cell Culture, Incubation, Flow Cytometry

Journal:

Article Title: MUC1 oncoprotein promotes autophagy in a survival response to glucose deprivation

doi:

Figure Lengend Snippet: MUC1 blocks ROS-induced necrosis in the response to glucose deprivation. (A and B) HCT116/vector and HCT116/MUC1 cells were cultured in 25, 1 or 1 mM glucose and 500 U/ml catalase for 24 h. The cells were stained with PI and analyzed by flow cytometry (A). The results obtained with the HCT116/vector cells cultured in 25 mM glucose (solid bars), 1 mM glucose (open bars) or 1 mM glucose and catalase (shaded bars) are expressed as the percentage (mean ± SD of three experiments) necrotic cells (B). (C and D) HCT116/vector and HCT116/MUC1 cells were cultured in 25, 1 or 1 mM glucose and 10 mM NAC for 24 h. The cells were stained with PI and analyzed by flow cytometry (C). The results obtained with the indicated cells cultured in 25 mM glucose (solid bars), 1 mM glucose (open bars) or 1 mM glucose and NAC (shaded bars) are expressed as the percentage (mean ± SD of three experiments) necrotic cells (D).

Article Snippet: Cells were lysed as described ( 46 ) and analyzed by immunoblotting with anti-MUC1-C (Ab5; NeoMarkers Inc., Fremont, CA), anti-β-actin (Sigma), anti-MAP1LC3 (NanoTools; Freiburg, Germany), anti-phospho-Akt (anti-p-Akt) anti-Akt, anti-phospho-AMPKα (Thr-172) (p-AMPK) and anti-AMPKα (Cell Signaling Inc.).

Techniques: Plasmid Preparation, Cell Culture, Staining, Flow Cytometry

Journal:

Article Title: MUC1 oncoprotein promotes autophagy in a survival response to glucose deprivation

doi:

Figure Lengend Snippet: MUC1 promotes autophagy in the response to glucose deprivation. (A and B) HCT116/MUC1 cells were cultured in 1 mM glucose and the absence (solid bars and squares) or presence (open bars and squares) of 10 mM 3-MA for 0–72 h. Intracellular ATP levels are expressed as mean ± SD of three separate experiments relative to that at 0 h (A). Cell number as assessed by trypan blue staining is expressed as the mean ± SD of three separate experiments (B). (C and D) HCT116/MUC1 cells were transfected with a non-specific (NS) siRNA or ATG7 siRNA for 5 days. Total cellular RNA was amplified with ATG7- and β-actin-specific primers (C). Intracellular ATP levels for ATG7 siRNA transfected cells (open bars) relative to that obtained with the NS siRNA transfected cells (solid bars) are expressed as mean ± SD of three separate experiments (D, left). Numbers of NS siRNA (solid bars) and ATG7 siRNA (open bars) transfected cells as assessed by trypan-blue staining is expressed as the mean ± SD of three separate experiments (D, right). (E) HCT116/vector and HCT116/MUC1 cells were cultured in 1 mM glucose for 24 h in the absence and presence of 50 μM CQ for the last 10 h. Lysates were immunoblotted with the indicated antibodies.

Article Snippet: Cells were lysed as described ( 46 ) and analyzed by immunoblotting with anti-MUC1-C (Ab5; NeoMarkers Inc., Fremont, CA), anti-β-actin (Sigma), anti-MAP1LC3 (NanoTools; Freiburg, Germany), anti-phospho-Akt (anti-p-Akt) anti-Akt, anti-phospho-AMPKα (Thr-172) (p-AMPK) and anti-AMPKα (Cell Signaling Inc.).

Techniques: Cell Culture, Staining, Transfection, Amplification, Plasmid Preparation

Journal:

Article Title: MUC1 oncoprotein promotes autophagy in a survival response to glucose deprivation

doi:

Figure Lengend Snippet: MUC1 promotes autophagy in response to glucose deprivation by an AMPK-dependent mechanism. (A and B) HCT116/vector-A and HCT116/MUC1-A cells were cultured in the presence of 25 or 1 mM glucose for the indicated times. Lysates were immunoblotted with the indicated antibodies. (C and D) HCT116/MUC1 cells were cultured in 1 mM glucose in the absence or presence of 10 μM compound C for 24 h. CQ was added as indicated during the last 10 h. Lysates were immunoblotted with the indicated antibodies.

Article Snippet: Cells were lysed as described ( 46 ) and analyzed by immunoblotting with anti-MUC1-C (Ab5; NeoMarkers Inc., Fremont, CA), anti-β-actin (Sigma), anti-MAP1LC3 (NanoTools; Freiburg, Germany), anti-phospho-Akt (anti-p-Akt) anti-Akt, anti-phospho-AMPKα (Thr-172) (p-AMPK) and anti-AMPKα (Cell Signaling Inc.).

Techniques: Plasmid Preparation, Cell Culture