Journal: Cell

Article Title: A Genome-wide ER-Phagy Screen Highlights Key Roles of Mitochondrial Metabolism and ER-Resident UFMylation

doi: 10.1016/j.cell.2020.02.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The blots were scanned using Li-Cor’s Near-InfraRed fluorescence Odyssey CLx Imaging System, and densitometry quantifications were done using Li-Cor’s ImageStudio software complementary of Odyssey.

Techniques: Recombinant, Viability Assay, Magnetic Beads, Genome Wide, Sequencing, CRISPR, Software

Journal: Cell Reports Methods

Article Title: RECOVER identifies synergistic drug combinations in vitro through sequential model optimization

doi: 10.1016/j.crmeth.2023.100599

Figure Lengend Snippet:

Article Snippet: Felbamate , MedChem Express , HY-B0184.

Techniques: Software, Recombinant

Journal: Theranostics

Article Title: Abnormal mitochondrial iron metabolism damages alveolar type II epithelial cells involved in bleomycin-induced pulmonary fibrosis

doi: 10.7150/thno.94072

Figure Lengend Snippet: MFRN2 promoted mitochondrial iron deposition in single-dose BLM-induced pulmonary fibrosis in mice and BLM-induced damage in MLE-12 cells. (A-B) MFRN2 protein levels in the lungs was detected using western blotting ( n = 3). (C) MFRN2 localization in AECII from control and pulmonary fibrosis mice was detected using anti-SPC antibodies (green) and anti-MFRN2 antibodies (red) (Scale bar = 50 μm). (D-E) MFRN2 protein levels in MLE-12 cells was detected using western blotting ( n = 3). (F) MFRN2 localization in the mitochondria of control and BLM-injured MLE-12 cells was determined using anti-MFRN2 antibodies (green) and Mito-Tracker (red) (Scale bar = 100 μm). (G) MFRN2 protein levels in MLE-12 cells was detected using western blotting ( n = 3). (H) MLE-12 cells were stained with Mito-Tracker (red) and Mito-Ferro Green (green) and then imaged using confocal microscopy with Airyscan. Representative images are shown (Scale bar = 5 μm), and boxes mark the enlarged images shown (Scale bar =1 μm). (I) The two mitochondrial DNA fragments Cytb-Nd1 and COX III-Cytb were detected using agarose gel electrophoresis. (J) MLE-12 cell viability was analyzed using CCK-8 ( n = 5). (K) MFRN2 overexpressing mitochondria were extracted was cocultured with healthy MLE-12 cells. (L) MLE-12 cells were stained with Mito-Tracker (red), and the iron was stained with Mito-Ferro Green (green). Representative images are shown (Scale bar = 5 μm), and boxes mark the enlarged images shown (Scale bar = 1 μm). (M) The Δψm in MLE-12 cells was measured using JC-1 staining (Scale bar = 100 μm). (N) MLE-12 cell viability was analyzed using CCK-8 ( n = 9). * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: The procedure was performed with primary antibodies: Collagen I (Cat# 14695-1-AP; Proteintech, China; 1:2000), Collagen III (Cat# 22734-1-AP; Proteintech, China; 1:2000), α-SMA (Cat# 19245; Cell Signaling Technology, USA; 1:2000), FN (Cat# 15613-1-AP; Proteintech, China; 1:2000), β-Tubulin (Cat# 10094-1-AP; Proteintech, China; 1:10000), TFAM (Cat# 22586-1-AP; Proteintech, China; 1:2000), MFRN2 (Cat# orb457153; Biorbyt; United Kingdom; 1:2000), FBXL5 (Cat# A5602; ABclonal; USA; 1:100), IREB2 (Cat# 23829-1-AP; Proteintech, China; 1:2000), Ubiquitin (Cat# 10201-2-AP; Proteintech, China; 1:2000).

Techniques: Western Blot, Control, Staining, Confocal Microscopy, Agarose Gel Electrophoresis, CCK-8 Assay

Journal: Theranostics

Article Title: Abnormal mitochondrial iron metabolism damages alveolar type II epithelial cells involved in bleomycin-induced pulmonary fibrosis

doi: 10.7150/thno.94072

Figure Lengend Snippet: FBXL5 regulated the IREB2-MFRN2 axis, attenuating mitochondrial iron deposition and protecting AECII cells from single-dose BLM-induced injury and MLE-12 cells from BLM damage. (A-B) IREB2 protein levels in the lungs of mice was detected using western blotting ( n = 3). (A and C) IREB2 protein levels in MLE-12 cells was detected using western blotting ( n = 3). (D) IREB2 localization in AECII of the control and pulmonary fibrosis mice was determined using anti-IREB2 antibodies (green) and anti-SPC antibodies (red) (Scale bar = 50 μm). (E) IREB2 protein levels in MLE-12 cells was detected using immunofluorescence staining (Scale bar = 100 μm). (F) The interaction between IREB2 and MFRN2 was assessed by dual luciferase reporter gene assay ( n = 3). (G-J) FBXL5, IREB2, and MFRN2 protein levels in MLE-12 cells was detected using western blotting ( n = 3). (K) MLE-12 cells were stained with Mito-Tracker (red), and iron was stained with Mito-Ferro Green (green), and then they were imaged using confocal microscopy with Airyscan. Representative images are shown (Scale bar = 5 μm), and boxes mark the enlarged images shown (Scale bar = 1 μm). (L) TFAM protein levels in MLE-12 cells was detected using immunofluorescence staining (Scale bar = 100 μm). (M) MLE-12 cells were transduced with Mito-Tracker (red), and the DNA was transduced with anti-DNA antibody (green) and then imaged by confocal microscopy with Airyscan. Representative images are shown (Scale bar = 1 μm), and boxes mark the enlarged images shown (Scale bar = 1 μm). (N) The Δψm in MLE-12 cells was measured using JC-1 staining (Scale bar = 100 μm). * P < 0.05, ** P < 0.01.

Article Snippet: The procedure was performed with primary antibodies: Collagen I (Cat# 14695-1-AP; Proteintech, China; 1:2000), Collagen III (Cat# 22734-1-AP; Proteintech, China; 1:2000), α-SMA (Cat# 19245; Cell Signaling Technology, USA; 1:2000), FN (Cat# 15613-1-AP; Proteintech, China; 1:2000), β-Tubulin (Cat# 10094-1-AP; Proteintech, China; 1:10000), TFAM (Cat# 22586-1-AP; Proteintech, China; 1:2000), MFRN2 (Cat# orb457153; Biorbyt; United Kingdom; 1:2000), FBXL5 (Cat# A5602; ABclonal; USA; 1:100), IREB2 (Cat# 23829-1-AP; Proteintech, China; 1:2000), Ubiquitin (Cat# 10201-2-AP; Proteintech, China; 1:2000).

Techniques: Western Blot, Control, Immunofluorescence, Staining, Luciferase, Reporter Gene Assay, Confocal Microscopy, Transduction

Journal: Theranostics

Article Title: Abnormal mitochondrial iron metabolism damages alveolar type II epithelial cells involved in bleomycin-induced pulmonary fibrosis

doi: 10.7150/thno.94072

Figure Lengend Snippet: Activation of the EP4 receptor improved mitochondrial iron deposition in MLE-12 cells, mitigating injury following BLM exposure. (A) MLE-12 cells were transduced with Mito-Tracker (red), and iron was transduced with Mito-Ferro Green (green) and then imaged using confocal microscopy with Airyscan. Representative images are shown (Scale bar = 5 μm), and boxes mark the enlarged images shown (Scale bar = 1 μm). (B) Mitochondrial iron content was analyzed using an iron kit ( n = 3). (C) The Δψm in MLE-12 cells was measured using JC-1 staining (Scale bar = 100 μm). (D) Oxygen consumption rate (OCR) analysis of MLE-12 (J, n = 3). (E) MLE-12 cell viability was analyzed using CCK-8 ( n = 10). (F-H) FBXL5 and MFRN2 protein levels in MLE-12 cells was detected using western blotting ( n = 3). (I) IREB2 protein levels in MLE-12 cells was detected using immunofluorescence staining (Scale bar = 100 μm). (J) MFRN2 protein levels in MLE-12 cells was detected using immunofluorescence staining (Scale bar = 100 μm). (K) IREB2 ubiquitination was analyzed using Co-IP. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The procedure was performed with primary antibodies: Collagen I (Cat# 14695-1-AP; Proteintech, China; 1:2000), Collagen III (Cat# 22734-1-AP; Proteintech, China; 1:2000), α-SMA (Cat# 19245; Cell Signaling Technology, USA; 1:2000), FN (Cat# 15613-1-AP; Proteintech, China; 1:2000), β-Tubulin (Cat# 10094-1-AP; Proteintech, China; 1:10000), TFAM (Cat# 22586-1-AP; Proteintech, China; 1:2000), MFRN2 (Cat# orb457153; Biorbyt; United Kingdom; 1:2000), FBXL5 (Cat# A5602; ABclonal; USA; 1:100), IREB2 (Cat# 23829-1-AP; Proteintech, China; 1:2000), Ubiquitin (Cat# 10201-2-AP; Proteintech, China; 1:2000).

Techniques: Activation Assay, Transduction, Confocal Microscopy, Staining, CCK-8 Assay, Western Blot, Immunofluorescence, Ubiquitin Proteomics, Co-Immunoprecipitation Assay

Journal: Theranostics

Article Title: Abnormal mitochondrial iron metabolism damages alveolar type II epithelial cells involved in bleomycin-induced pulmonary fibrosis

doi: 10.7150/thno.94072

Figure Lengend Snippet: Activation of the EP4 receptor improved BLM-induced mitochondrial iron deposition via FBXL5 regulation of the IREB2-MFRN2 axis in MLE-12 cells. (A-B) Ubiquitin, IREB2, and TFAM protein levels in MLE-12 cells was detected using western blotting ( n = 3). (C) MLE-12 cell viability was analyzed using CCK-8 ( n = 5). (D-E) FBXL5, IREB2, and MFRN2 protein levels in MLE-12 cells was detected using western blotting ( n = 3). (F) MLE-12 cell viability was analyzed using CCK8 ( n = 5). (G) MLE-12 cells were transduced with Mito-Tracker (red), and iron was transduced with Mito-Ferro Green (green) and then imaged using confocal microscopy with Airyscan. Representative images are shown (Scale bar = 5 μm), and boxes mark the enlarged images as shown (Scale bar = 1 μm). (H) MLE-12 cells were transduced with Mito-Tracker (red), and DNA was transduced with anti-DNA antibodies (green) and then imaged using a confocal microscopy with Airyscan. Representative images are shown (Scale bar = 5 μm), and boxes mark the enlarged images shown (Scale bar = 1 μm). (I) ROS levels were analyzed using a ROS kit (Scale bar = 100 μm). (J) The Δψm in MLE-12 cells was measured using JC-1 staining (Scale bar = 100 μm). (K) MLE-12 cells were transduced with Mito-Tracker (red), and iron was transduced with Mito-Ferro Green (green) and then imaged using confocal microscopy with Airyscan. Representative images are shown (Scale bar = 5 μm), and boxes mark the enlarged images shown (Scale bar = 1 μm). (L) MLE-12 cells were transduced with Mito-Tracker (red), and DNA was transduced with anti-DNA antibodies (green) and then imaged using confocal microscopy with Airyscan. Representative images are shown (Scale bar = 5 μm), and boxes mark the enlarged images shown (Scale bar =1 μm). (M) ROS levels were analyzed using an ROS kit (Scale bar = 100 μm). (N) The Δψm in MLE-12 cells was measured using JC-1 staining (Scale bar = 100 μm). * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: The procedure was performed with primary antibodies: Collagen I (Cat# 14695-1-AP; Proteintech, China; 1:2000), Collagen III (Cat# 22734-1-AP; Proteintech, China; 1:2000), α-SMA (Cat# 19245; Cell Signaling Technology, USA; 1:2000), FN (Cat# 15613-1-AP; Proteintech, China; 1:2000), β-Tubulin (Cat# 10094-1-AP; Proteintech, China; 1:10000), TFAM (Cat# 22586-1-AP; Proteintech, China; 1:2000), MFRN2 (Cat# orb457153; Biorbyt; United Kingdom; 1:2000), FBXL5 (Cat# A5602; ABclonal; USA; 1:100), IREB2 (Cat# 23829-1-AP; Proteintech, China; 1:2000), Ubiquitin (Cat# 10201-2-AP; Proteintech, China; 1:2000).

Techniques: Activation Assay, Ubiquitin Proteomics, Western Blot, CCK-8 Assay, Transduction, Confocal Microscopy, Staining

Journal: Theranostics

Article Title: Abnormal mitochondrial iron metabolism damages alveolar type II epithelial cells involved in bleomycin-induced pulmonary fibrosis

doi: 10.7150/thno.94072

Figure Lengend Snippet: Activation of EP4 receptor mitigated AECII mitochondrial iron deposition via FBXL5 regulation of the IREB2-MFRN2 axis in single-dose BLM-induced pulmonary fibrosis. (A) mRNA levels of Ptger1 , Ptger2, and Ptger4 in the lungs was detected using RT-qPCR ( n =5). (B) EP1 localization in AECII from control and pulmonary fibrosis mice was determined using anti-EP1 antibodies (green) and anti-SPC antibodies (red) (Scale bar = 40 μm). (C) EP2 localization in AECII from control and pulmonary fibrosis mice was determined using anti-EP2 antibodies (green) and anti-SPC antibodies (red) (Scale bar = 40 μm). (D) EP4 localization in AECII from control and pulmonary fibrosis mice was determined using anti-EP4 antibodies (green) and anti-SPC antibodies (red) (Scale bar = 40 μm). (E) Lung histopathological analysis was performed using H&E staining (Scale bar = 100 μm). (F) Masson's staining was employed to evaluate collagen disposition (Scale bar = 100 μm). (G) mRNA levels of Acta2 , Col1a1, and Col3a1 in the lungs was detected using RT-qPCR ( n =5). (H-I) α-SMA, Collagen I, Collagen III, and SPC protein levels in the lung was detected using western blotting ( n = 3). (J) Iron deposition in lung tissues was assessed using Prussian blue staining (Scale bar = 50 μm). (K-L) MFRN2, FBXL5, IREB2, and TFAM protein levels in the lungs was detected using western blotting ( n = 3). (M) Confocal detection of mitochondrial iron deposition in primary AECII (Scale bar = 3 μm). * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: The procedure was performed with primary antibodies: Collagen I (Cat# 14695-1-AP; Proteintech, China; 1:2000), Collagen III (Cat# 22734-1-AP; Proteintech, China; 1:2000), α-SMA (Cat# 19245; Cell Signaling Technology, USA; 1:2000), FN (Cat# 15613-1-AP; Proteintech, China; 1:2000), β-Tubulin (Cat# 10094-1-AP; Proteintech, China; 1:10000), TFAM (Cat# 22586-1-AP; Proteintech, China; 1:2000), MFRN2 (Cat# orb457153; Biorbyt; United Kingdom; 1:2000), FBXL5 (Cat# A5602; ABclonal; USA; 1:100), IREB2 (Cat# 23829-1-AP; Proteintech, China; 1:2000), Ubiquitin (Cat# 10201-2-AP; Proteintech, China; 1:2000).

Techniques: Activation Assay, Quantitative RT-PCR, Control, Staining, Western Blot

Journal: Theranostics

Article Title: Abnormal mitochondrial iron metabolism damages alveolar type II epithelial cells involved in bleomycin-induced pulmonary fibrosis

doi: 10.7150/thno.94072

Figure Lengend Snippet: Sequences of specific primers used in this study.

Article Snippet: The procedure was performed with primary antibodies: Collagen I (Cat# 14695-1-AP; Proteintech, China; 1:2000), Collagen III (Cat# 22734-1-AP; Proteintech, China; 1:2000), α-SMA (Cat# 19245; Cell Signaling Technology, USA; 1:2000), FN (Cat# 15613-1-AP; Proteintech, China; 1:2000), β-Tubulin (Cat# 10094-1-AP; Proteintech, China; 1:10000), TFAM (Cat# 22586-1-AP; Proteintech, China; 1:2000), MFRN2 (Cat# orb457153; Biorbyt; United Kingdom; 1:2000), FBXL5 (Cat# A5602; ABclonal; USA; 1:100), IREB2 (Cat# 23829-1-AP; Proteintech, China; 1:2000), Ubiquitin (Cat# 10201-2-AP; Proteintech, China; 1:2000).

Techniques: Sequencing