Journal: eLife

Article Title: Ezrin defines TSC complex activation at endosomal compartments through EGFR–AKT signaling

doi: 10.7554/eLife.98523

Figure Lengend Snippet:

Article Snippet: Antibody , anti-LAMP1 (mouse monoclonal) , DSHB , H4A3 , IF (1:1000).

Techniques: CRISPR, Transfection, Construct, Sequencing, Recombinant, Purification, Western Blot, Software, Mass Spectrometry, Immunofluorescence, Functional Assay, Fluorescence, Imaging, Staining, Plasmid Preparation

Journal: Acta biochimica et biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2.

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Figure 2. Inhibition of miR-29b-1-5p ameliorates lipopolysaccharide (LPS)-induced septic myocardial injury in mice (A) Mice received a single intraperitoneal injection of 25 mg/kg LPS (lethal dose) or a tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving LPS injection), after which the survival conditions were assessed for 96 h, and survival rate curves were generated. An equal amount of saline was injected into the control group. n=20. Mice received a single intraperitoneal injection of 10 mg/kg LPS or a tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving LPS injection). (B) Twenty-four hours after LPS injection, echocardiography tests were performed. (C,D) Mice were euthanized, and histological analysis of myocardial tissue injury was performed via hematoxylin and eosin (HE) staining and Masson’s trichrome staining. Scale bar: 50 μm. (F,G) The cardiac injury score and fibrotic area were assessed via HE and Masson’s trichrome staining, respectively. (E,H) α-Smooth muscle actin (α-SMA) expression in mouse myocardial tissues was determined via immunohistochemistry (IHC) staining. Scale bar: 20 μm. (I‒L) Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and cardiac troponin I (cTnI) levels in mouse serum were determined by enzyme-linked immunosorbent assay (ELISA). (M) The miR-29b-1-5p expression level in mouse myocardial tissue was determined by real-time quantitative reverse transcription PCR (qRT-PCR). n=6. (N) The protein levels of TLR2, TLR4, TFEB, LAMP1 and LC3II in mouse myocardial tissues were determined by western blot analysis. n=3. **P<0.01 vs the control group; #P<0.05 and ##P<0.01 vs. the LPS+NC antagomir group.

Article Snippet: Jiang et al. Acta Biochim Biophys Sin 2024 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Injection, Generated, Saline, Control, Staining, Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Quantitative RT-PCR, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2.

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Figure 3. Inhibition of miR-29b-1-5p ameliorates CLP-induced septic myocardial injury in mice (A) Mice were subjected to CLP surgery or tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving CLP surgery), after which the survival conditions were assessed for 96 h, and survival rate curves were generated. n=20. A sham operation was performed on the control group. n=20. (B) Twenty- four hours after CLP surgery, echocardiography was performed. (C,D) Mice were euthanized, and histological analysis of myocardial tissue injury was performed via HE staining and Masson’s trichrome staining. Scale bar: 50 μm. (F,G) The cardiac injury score and fibrotic area were assessed via HE and Masson’s trichrome staining, respectively. (E,H) The protein levels and distribution of α-SMA in mouse myocardial tissues were examined using IHC staining. Scale bar: 20 μm. (I‒L) TNF-α, IL-1β, IL-6 and cTnI levels in mouse serum were determined by ELISA. (M) The miR-29b- 1-5p expression level in mouse myocardial tissue was determined by qRT-PCR. n=6. (N) The protein levels of TLR2, TLR4, TFEB, LAMP1 and LC3II in mouse myocardial tissues were determined by western blot analysis. n=3. **P<0.01 vs the control group; #P<0.05 and ##P<0.01 vs the CLP+NC antagomir group.

Article Snippet: Jiang et al. Acta Biochim Biophys Sin 2024 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Injection, Generated, Control, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2.

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Figure 4. Inhibition of miR-29b-1-5p ameliorates LPS-induced cardiomyocyte dysfunction in mice in vitro (A) Twenty-four hours after transfecting HL-1 cells with the NC or miR-29b-1-5p antagomir, LPS (1 μg/mL) stimulation was applied for another 24 h, and miR-29b-1-5p expression was validated in each group by qRT-PCR. (B) After LPS treatment for 0, 24, 48, or 72 h, MTT assay was performed to examine the viability of the HL-1 cells. (C) Flow cytometry was used to evaluate apoptosis in HL-1 cells. (D‒F) TNF-α, IL-1β, and IL-6 levels in HL-1 cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA). (G‒I) Western blot analysis was used to measure Bax, Bcl-2, cleaved caspase-3, pro- caspase-3, TLR2, TLR4, TFEB, LAMP1 and LC3II protein levels in HL-1 cells. n=3. **P<0.01 vs the control group; ##P<0.01 vs the LPS+NC antagomir group.

Article Snippet: Jiang et al. Acta Biochim Biophys Sin 2024 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, In Vitro, Expressing, Quantitative RT-PCR, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Acta biochimica et biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2.

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Figure 6. Silencing of TERF2 partially reverses the ameliorative effect of miR-29b-1-5p inhibition on LPS-induced cardiomyocyte apoptosis and inflammation (A) HL-1 cells were transfected with sh-TERF2 or sh-NC vector. Forty-eight hours later, HL-1 cells were collected to detect the protein expression of TERF2. (B‒I) HL-1 cells were transfected with sh-TERF2 or the miR-29b-1-5p antagomir for 24 h, followed by stimulation with LPS. MTT assay was performed to examine HL-1 cell viability (B). Flow cytometry was performed to evaluate HL-1 cell apoptosis (C). ELISA was also conducted to evaluate TNF-α, IL-1β, and IL-6 levels in HL-1 cells (D‒F). Western blot analysis was conducted to determine the protein levels of TERF2, cleaved PARP, cleaved caspase-3, pro-caspase-3, TLR2, TLR4, TFEB, LAMP1 and LC3II in HL-1 cells (G–I). **P<0.01 vs LPS+NC antagomir+ sh-NC; #P<0.05, ##P<0.01 vs LPS+miR-29b-1-5p antagomir+sh-TERF2.

Article Snippet: Jiang et al. Acta Biochim Biophys Sin 2024 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Talin2 mediates secretion and trafficking of matrix metallopeptidase 9 during invadopodium formation.

doi: 10.1016/j.bbamcr.2020.118693

Figure Lengend Snippet: Fig. 4. Talin2-KO caused an accumulation of enlarged MMP9 vesicles. A. Number of enlarged vesicles per cell in talin2-knockout cells, control cells, and control cells treated for 6 h with 200 μM chloroquine (CQ) (Control, n = 41; TLN2-KO#1, n = 47; TLN2-KO#2, n = 48; Control + CQ, n = 12). Comparison between cells treated with chloroquine and KO cells shows no statistical difference (P > 0.05) U test, ***P < 0.001. B. Pie diagrams showing percentage of cells having a) five or more enlarged vesicles (diameter of 1 μm or more), b) having at least one vesicle with diameter of > 2 μm, c) fulfilling condition a and b, and d) the rest of the cells. C. MMP9 was co-localized with LAMP1 in MDA-MB-231 cells plated on fibronectin after treatment with lysosome inhibitors: 0.2 μM Bafilomycin (Baf) or 200 μM Chloroquine (CQ) for 6 h. In both cases MMP9 was accumulated in multiple enlarged vesicles. Cells were transfected with MMP9-EGFP and stained with anti-LAMP1 antibody. Representative maximum intensity projections (MIP) of confocal images are shown.

Article Snippet: Anti-EEA1 rabbit monoclonal antibody (C45B10) and anti-LAMP1 mouse monoclonal antibody (D4O1S) were from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Knock-Out, Control, Comparison, Transfection, Staining

Journal: Scientific Reports

Article Title: Analysis of the intracellular traffic of IgG in the context of Down syndrome (trisomy 21)

doi: 10.1038/s41598-021-90469-z

Figure Lengend Snippet: Characterization of the compartments involved in the trafficking of IgG. ( A ) Representative confocal microscopy images showing the distribution of early endosomes (EEA1, cyan), recycling endosomes (Rab11, green), and lysosomes (LAMP1, orange) in diploid (NDS) and trisomic (DS) fibroblasts after incubation with IgG for 60 min at 37 °C. Scale bar: 10 µm. ( B ) Number and size of EEA1 + endosomes (upper panel), Rab11 + recycling endosomes (upper middle panel), and lysosomes (LAMP1, Lysotracker) (lower middle and lower panels) in NDS (NDS-1, -2, and -3) and DS (DS-1, -2, and -3) cells. Each bar represents the mean ± SD. Each point represents measurements in individual cells (NDS, 72–296 cells/marker; DS, 82–250 cells cells/marker). **P < 0.01, ***P < 0.001, ns = not significant, Student’s t test.

Article Snippet: Samples were blocked in 3% bovine serum albumin (BSA)-PBS for 1 h, and then incubated for 2 h at room temperature with the following primary antibodies: rabbit anti-EEA1 (1:3000, Thermo Fisher Scientific Cat# PA1-063A, RRID:AB_2096819), rabbit anti-Rab11 (1:50, Thermo Fisher Scientific Cat# 71-5300, RRID:AB_2533987), and mouse anti-LAMP1 (1:100, Thermo Fisher Scientific Cat# 14-1079-80, RRID:AB_467426).

Techniques: Confocal Microscopy, Incubation, Marker

Journal: Scientific Reports

Article Title: Analysis of the intracellular traffic of IgG in the context of Down syndrome (trisomy 21)

doi: 10.1038/s41598-021-90469-z

Figure Lengend Snippet: Subcellular distribution of FcRn in trisomic cells. ( A ) Localization of GFP-tagged FcRn (green) in early endosomes (EEA1, red) and lysosomes (LAMP1, magenta) in representative diploid (NDS) and trisomic (DS) fibroblasts. Scale bar: 10 µm. ( B ) FcRn + vesicles number and size. ( C ) Quantitative colocalization analysis of FcRn with EEA1 + endosomes (left panel) or LAMP1 + lysosomes (right panel). Each bar represents the mean ± SD. Each point represents measurements in individual cells (NDS-1, -2, and -3 cell lines, n = 53 cells; DS-1, -2, and -3 cell lines, n = 58 cells). *P < 0.05, ***P < 0.001, ns = not significant, Student’s t test.

Article Snippet: Samples were blocked in 3% bovine serum albumin (BSA)-PBS for 1 h, and then incubated for 2 h at room temperature with the following primary antibodies: rabbit anti-EEA1 (1:3000, Thermo Fisher Scientific Cat# PA1-063A, RRID:AB_2096819), rabbit anti-Rab11 (1:50, Thermo Fisher Scientific Cat# 71-5300, RRID:AB_2533987), and mouse anti-LAMP1 (1:100, Thermo Fisher Scientific Cat# 14-1079-80, RRID:AB_467426).

Techniques:

Journal: Journal of Lipid Research

Article Title: Role of N -glycosylation of human lysosomal phospholipase A2 for the formation of catalytically active enzyme

doi: 10.1194/jlr.M041640

Figure Lengend Snippet: Subcellular fractionation of WT and mutated hLPLA2 gene transfectants by differential centrifugation. All manipulations in the subcellular fractionation were carried out at 4°C. One milliliter of cell homogenate (1 mg protein/ml) obtained from WT, N1A, or Nt gene transfectants was centrifuged for 10 min at 600 g. The resultant supernatant and pellet were collected as post nuclear supernatant (PNS) and nuclear fraction, respectively. The PNS was centrifuged for 10 min at 15,000 g. The resultant pellets were collected as crude mitochondrial fraction (MIT). The supernatant obtained at 15,000 g was centrifuged for 1.5 h at 160,000 g. The resultant supernatant and pellet were collected as soluble fraction (SOL) and microsomal fraction (MIC), respectively. The pellet of MIT and MIC was dispersed with 1 ml of 0.25 M sucrose, 10 mM HEPES (pH 7.4), and 1 mM EDTA. Twenty microliters of the cell homogenate (HOM), PNS, MIT, and MIC suspensions, and SOL was used to assure the distribution of LPLA2 in the cell. SDS polyacrylamide gel electrophoresis and Western blotting were carried out as described in the Materials and Methods. In (A), distribution of WT in the cell was examined by treatment of the membrane with anti-hLPLA2 antibody (LP) or anti-lamp1 antibody (LM). In (B), N1A and Nt were found in MIC. The left and middle membranes were treated with anti-hLPLA2 antibody. The right membrane was treated with anti-calnexin antibody.

Article Snippet: Reagents 1,2-Dioleoyl- sn -glycero-3-phosphocholine (DOPC), sulfatide, N -acetylsphingosine (NAS), and N -oleoyl-sphingosine were obtained from Avanti Polar Lipids Corp. (Alabaster, AL); high performance thin layer chromatography silica gel plates, 10 × 20 cm, were from Merck (Darmstadt, Germany); N -glycosidase F (PNGase F) and POD immunostain set were from Wako (Tokyo, Japan); anti-rabbit IgG goat polyclonal antibody, HRP-conjugate, was from MP Biomedicals (Solon, OH); anti-lamp1 mouse monoclonal antibody and anti-calnexin rabbit polyclonal antibody were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Fractionation, Centrifugation, Polyacrylamide Gel Electrophoresis, Western Blot, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Vesicular Polyamine Transporter Mediates Vesicular Storage and Release of Polyamine from Mast Cells *

doi: 10.1074/jbc.M116.756197

Figure Lengend Snippet: Localization of VPAT in BMMCs. a–c, BMMCs were fixed and subjected to double immunostaining with antibodies to VPAT (green, left), histamine, serotonin, cathepsin D, and spermine synthase (a); VAMP2, VAMP3, VAMP7, and VAMP8 (b); and LAMP1 and GM130 (c) (red, middle). Merged images (right) are also shown. Areas surrounded by dotted lines are enlarged in insets. Arrowheads, merged regions. Bars, 10 μm.

Article Snippet: The following antibodies were obtained commercially: anti-histamine mouse monoclonal (Abcam, Cambridge, UK), anti-serotonin mouse monoclonal (Dako, Glostrup, Denmark), anti-cathepsin D goat polyclonal (Santa Cruz Biotechnology, Inc., Dallas, TX), anti-VAMP2 mouse monoclonal (Synaptic Systems, Göttingen, Germany), anti-VAMP3 sheep polyclonal (Abcam), anti-VAMP7 mouse monoclonal (Abcam), anti-VAMP8 mouse monoclonal (Santa Cruz Biotechnology), anti-LAMP1 mouse monoclonal (StressMarq Biosciences, Victoria, Canada), anti-GM130 mouse monoclonal (BD Biosciences), anti-spermine rabbit polyclonal (Novus Biologicals, Littleton, CO), and anti-spermidine rabbit polyclonal (Novus Biologicals) antibodies.

Techniques: Double Immunostaining