Journal: International Journal of Biological Sciences

Article Title: The Role of Autophagy in Lamellar Body Formation and Surfactant Production in Type 2 Alveolar Epithelial Cells

doi: 10.7150/ijbs.64285

Figure Lengend Snippet: Atg7-deficient mice show defects in lung development, LB formation, and surfactant production. (A) Gross appearance of representative neonates. The bodyweight of Atg7 -/- mice was slightly but significantly lower than that of wild-type (WT) mice (1.42g vs. 1.58g; n = 5). (B) Histological analysis of lungs from WT control (Ctrl) and Atg7 KO neonates (scale bar: 100 µm). (C) Septal thickness was measured at all points in each visual field (at least five random visual fields in high magnification per mouse were used for measurements; WT, n = 8, Atg7 KO, n = 6). (D) Frozen sections of Ctrl and Atg7 KO neonate lungs were stained with the anti-ABCA3 antibody to detect LBs (scale bar: 5 µm). Representative single cells are shown. The average area (E) and size distribution (F) of LBs were quantified by ImageJ software (80 LBs/mouse in at least five random high magnification fields were measured; WT, n = 5; Atg7 KO, n = 3). (G) The average number of LBs per cell was quantified (at least 25 AT2 cells/mouse were quantified; WT, n = 5; Atg7 KO, n = 3). (H) Electron microscopy analysis shows a marked reduction in the quantity and quality of LBs in Atg7 KO mice compared with controls (* denotes LB; Nu denotes nucleus; Insets are enlarged LBs; scale bar: 1 µm). (I) Quantification of the average area of LBs (n = 4 mice/group). (J) The number of LBs per AT2 cell in each genotype (n = 4 mice/group). (K) The levels of SFTPB and pro-SFTPC in extracts of whole lung tissue from Ctrl and Atg7 KO mice were determined by western blotting. Experiments were repeated more than three times; representative images are shown. (L) Densitometry analysis of SFTPB, pro-SFTPC, p62, and LC3B II/I in Ctrl and Atg7 KO mice (for SFTPB, WT, n = 16, Atg7 KO, n = 13; for pro-SFTPC, p62, and LC3B II/I, WT, n = 9, Atg7 KO, n = 4). (M) Knockdown of ATG7 by adenoviral infection inhibited LB formation. Cells infected with ad-vector have both lysotracker staining and GFP-fluorescence; cells infected with ad-sh- ATG7 have GFP-fluorescence but lack lysotracker staining (scale bar: 5 μm). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Following permeabilization, sections were blocked with 10% normal horse serum in PBS supplemented with 0.1% Triton X-100 for 1 h at room temperature and then incubated overnight at 4 °C with rabbit polyclonal anti-SFTPB (1:2000, #07-614, Millipore), mouse polyclonal anti-ABCA3 (1:200, #AB24751, Abcam), anti-pro-SFTPC (1:200, #3786, Millipore) or LC3B (1:200, #18725-1-AP, Proteintech).

Techniques: Staining, Software, Electron Microscopy, Western Blot, Infection, Plasmid Preparation, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: The Role of Autophagy in Lamellar Body Formation and Surfactant Production in Type 2 Alveolar Epithelial Cells

doi: 10.7150/ijbs.64285

Figure Lengend Snippet: Lung dysplasia and abnormal LBs are observed in bronchioalveolar epithelium-specific Atg7 conditional knockout mice. (A) Histological analysis of neonatal lungs from Atg7 flox/+ , Atg7 flox/+ :(tetO)7-Cre, Atg7 flox/+ :SFTPC-rtTA, and Atg7 SPC neonates (scale bar: 100 µm). (B) Septal thickness was measured at all points in each visual field, and five visual fields for each animal were used for the measurements (n ≥3 mice/group). (C) Frozen sections of Atg7 flox/+ , Atg7 flox/+ :(tetO)7-Cre, Atg7 flox/+ :SFTPC-rtTA, and Atg7 SPC neonates' lungs were stained using anti-ABCA3 antibody to detect LBs (scale bar: 2.5 µm). (D) The average size of LBs in control and Atg7 SPC neonate lungs (≥80 LBs in at least 5 ransom high magnification fields were measured/mouse, mice/group n = 3). (E) EM analysis shows robust LBs in the control mice, whereas those in Atg7 SPC mice contain few lamellae (scale bar: 1 µm). (F) Western blot analysis of protein lysates prepared from the lungs of Atg7 SPC mice and littermate controls at P1. In Atg7 SPC neonates, LC3B-I failed to convert to LC3B-II, and p62 was accumulated. All experiments were performed at least three times; representative data are shown. A quantitative analysis of SFTPB (G) and pro-SFTPC (H) (mice/group n ≥ 4). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Following permeabilization, sections were blocked with 10% normal horse serum in PBS supplemented with 0.1% Triton X-100 for 1 h at room temperature and then incubated overnight at 4 °C with rabbit polyclonal anti-SFTPB (1:2000, #07-614, Millipore), mouse polyclonal anti-ABCA3 (1:200, #AB24751, Abcam), anti-pro-SFTPC (1:200, #3786, Millipore) or LC3B (1:200, #18725-1-AP, Proteintech).

Techniques: Knock-Out, Staining, Western Blot

Journal: PLoS ONE

Article Title: Induction of Cell Death in Growing Human T-Cells and Cell Survival in Resting Cells in Response to the Human T-Cell Leukemia Virus Type 1 Tax

doi: 10.1371/journal.pone.0148217

Figure Lengend Snippet: (A) Growing Kit 225 cells were transfected with RelA-specific siRNA and cultured for 72 h. RelA expression was examined by RT-PCR. GAPDH was used as an internal control. (B) Growing Kit 225 cells were transfected with RelA-specific siRNA 24 h before adenovirus infection (Ad-Tax1 or Ad-Con), and harvested 48 h and 72 h post infection for western blotting. RelA, p100, p52 and Tax1 expression was monitored by immunoblotting with anti-RelA, anti-p52 and anti-Tax1 antibodies. β-Tubulin was used as an internal control. (C) siRNA-treated growing or resting Kit 225 cells were infected with Ad-Tax1 or Ad-Con, and cultured for indicated times. Mitochondrial activity was measured by MTT assay. Relative percentages of Ad-Tax1 to Ad-Con are shown. *, p <0.05. (D) siRNA-treated growing Kit 225 cells were infected with Ad-Tax1 or Ad-Con, and cultured for 72 h. Tax1 expression and DNA fragmentation were measured by flow cytometry. (E) Percentage average number of cells undergoing apoptosis was calculated from three independent experiments. Values are shown as the means ± SE. *, p < 0.05.

Article Snippet: Anti-RelA (sc-372), anti-p52 (sc-3786), anti-p38α (sc-535), anti-JNK (sc-571), anti-nucleolin (sc-13057) and β-tubulin (sc-9104) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Infection, Western Blot, Activity Assay, MTT Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: Induction of Cell Death in Growing Human T-Cells and Cell Survival in Resting Cells in Response to the Human T-Cell Leukemia Virus Type 1 Tax

doi: 10.1371/journal.pone.0148217

Figure Lengend Snippet: (A) Kit 225 cells were transfected with p100-specific siRNA and cultured for 48 h. p100 expression was detected by western blotting. β-Tubulin was used as an internal control. (B) Growing Kit 225 cells were transfected with p100-specific siRNA and cultured for 24 h, and infected with Ad-Con or Ad-Tax1, followed by harvest at indicated times. Mitochondrial activity was measured by MTT assay. Relative values of Ad-Tax1 to Ad-Con are shown. *, p < 0.05. (C) Expression of adenovirus-derived Tax1 and its mutant Tax225–232 proteins was measured by immunoblotting with anti-Tax1 antibody (Taxy-7). p100 and p52 levels were detected by anti-p52 antibody, which recognizes both p100 and p52. β-Tubulin was used as an internal control. (D) Growing Kit 225 cells were infected with Ad-Tax1, its mutant Ad-Tax225–232 or Ad-Con, and cultured for indicated times. Mitochondrial activity was measured by MTT assay. Relative percentages of Ad-Tax1 or Ad-Tax225–232 samples to Ad-Con samples are shown. *, p < 0.05. (E) Growing Kit 225 cells were infected with Ad-Tax1 or Ad-Tax225–232, and cultured for 72 h. DNA fragmentation was measured by flow cytometry. The thick line and gray area indicate Ad-Tax1- or Ad-Tax225–232-treated cells and Ad-Con-treated cells, respectively.

Article Snippet: Anti-RelA (sc-372), anti-p52 (sc-3786), anti-p38α (sc-535), anti-JNK (sc-571), anti-nucleolin (sc-13057) and β-tubulin (sc-9104) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Cell Culture, Expressing, Western Blot, Control, Infection, Activity Assay, MTT Assay, Derivative Assay, Mutagenesis, Flow Cytometry