Journal: Journal of Nanobiotechnology

Article Title: Promoting collateral formation in type 2 diabetes mellitus using ultra-small nanodots with autophagy activation and ROS scavenging

doi: 10.1186/s12951-024-02357-z

Figure Lengend Snippet: MoS 2 NDs reversed the inhibition of endothelial autophagy induced by HG/Hypo.( A ) GSEA plot and heatmap of genes related to autophagy. ( B ) Representative images of a single cell expressing RFP-GFP-LC3 following different treatments. ( C ) The expressions of autophagy-related factors: LC3, p62, Lamp1. ( D ) Quantification of protein expression levels with different treatments, n = 3, and P values were calculated by two-way ANOVA with Tukey’s post hoc test. ( E ) EdU staining, ( F ) wound healing analysis, ( G ) Transwell analysis, ( H ) tube formation analysis, ( I ) Annexin-V/PI flow cytometric detection, and ( J ) JC-1 staining of HUVECs under HG/Hypo conditions treated with MoS 2 NDs with or without the autophagy inhibitor, Baf A1, n = 3, and P values were calculated by t -test. All data are expressed as mean ± SD.

Article Snippet: HUVECs were incubated with Baf A1 (1 nM) (Cat# HY-100558, MedChemExpress, USA), 3-MA (Cat#HY-19132, MedChemExpress, USA) and SQ22536 (250 µM) (Cat# S8283, Selleck, USA) to inhibit autophagy and cAMP level, respectively.

Techniques: Inhibition, Expressing, Staining

Journal: EMBO Reports

Article Title: Multinucleation resets human macrophages for specialized functions at the expense of their identity

doi: 10.15252/embr.202256310

Figure Lengend Snippet: Venn diagram showing the commonly upregulated transcripts ( n = 66) in LGCs, FBGCs and osteoclasts (in gray). KEGG and BioPlanet 2019 pathway analyses on the 66 commonly upregulated genes. Pathways in red are connected. Macrophage fusion and multinucleation causes an increased lysosomal compartment and cell metabolism. The commonly upregulated genes in LGCs, FBGCs and osteoclasts ( ATP6V1H , ATP6V1D , TFRC and SLC11A2 ) and their role in the lysosome‐iron pathway. The effect of lysosomotropic agents (hydroxychloroquine, hydroxy S; ammonium chloride, NH 4 Cl) and v‐ATPase inhibitors (Bafilomycin A1, Baf A1; Concanamycin A, Con A) on fusion and multinucleation in LGCs (upper panel), FBGCs (middle panel) and osteoclasts (lower panel). To test the effect of lysosome dysfunction on cellular iron, FeCl 3 was supplemented. Error bars are median with interquartile range; significance tested by one‐way Anova followed by Sídák's multiple comparisons on log transformed data; n = 4 donors; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Simultaneously, cells were incubated with either Bafilomycin A1 (Baf A1, Sanbio B.V., Cat # 11038‐500), Concanamycin A (Con A, Sigma‐Aldrich, Cat # 27689), Hydroxychloroquine sulfate (Hydroxy Sulfate, Sigma‐Aldrich, Cat # 379409) and Ammonium chloride (NH4Cl, Sigma‐Aldrich, Cat # A9434).

Techniques: Transformation Assay

Journal: International Journal of Oncology

Article Title: Dual targets of lethal apoptosis and protective autophagy in liver cancer with periplocymarin elicit a limited therapeutic effect

doi: 10.3892/ijo.2023.5492

Figure Lengend Snippet: The pro-apoptosis effect of PPM might impair the function of autophagic lysosomes. (A) LC3 were assessed using western blotting. (B) Electron microscopy showed that PPM treatment led to autolysosome increase. Black arrows indicate the autolysosomes. (C) HepG2 cells were treated with PPM (100 nmol/l) and/or Baf A1 (50 nmol/l) for 24 h. LC3-II and p62/SQSTM1 protein expression levels were assessed using western blotting. (D) HepG2 cells were transfected with RFP-GFP-LC3 lentivirus and treated with PPM (100 nmol/l) for 24 h. Cells were observed to distinguish between autophagosome (yellow puncta) after colocalization analysis using a confocal microscope. Scale bar=10 µ m. (E) HepG2 cells were treated with PPM (100 nmol/l) for 24 h and the protein expression levels of LAMP1, CTSB and CTSD were assessed using western blotting. (F) HepG2 cells were treated with PPM (100 nM) and/or 20 µ mol/l Z-VAD-FMK for 24 h. LC3 and p62/SQSTM1 levels were detected by using western blot analysis. GAPDH or β-actin were used as a loading control. Data are presented as mean ± SD, n=3; * P<0.05, ** P<0.01 and *** P<0.001 vs. control. &&& P<0.001 vs. 3-MA. $ P<0.05 vs. Baf A1. ## P<0.01 vs. PPM. n.s., not significant; ASS, autophagolysosome; M, mitochondria; PPM, periplocymarin; LC3, microtubule-associated protein light chain 3; SQSTM1/p62, sequestosome 1/p62; LAMP1, Lysosomal-associated membrane protein 1; CTSB, cathepsin B; CTSD, cathepsin D; RFP-GFP-LC3, red fluorescence point-green fluorescence point-LC3.

Article Snippet: HepG2 cells were treated with PPM in the presence or absence of 5 mM 3-methyladenine (3-MA, cat. no. HY-19312; MedChemExpress) , 50 nmol/l Bafilomycin A1 (Baf A1, cat. no. HY-100558; MedChemExpress) ( ) or 20 µ mol/l Z-VAD-FMK (cat. no. HY-16658B; MedChemExpress) ( ) at 37°C for 24 h. Total proteins were extracted from HepG2 cells using RIPA lysis buffer supplemented with Pierce Phosphatase Inhibitor (cat. no. A32961, Thermo Fisher Scientific, Inc.).

Techniques: Western Blot, Electron Microscopy, Expressing, Transfection, Microscopy, Control, Membrane, Fluorescence

Journal: Aging (Albany NY)

Article Title: PRMT5 facilitates angiogenesis and EMT via HIF-1α/VEGFR/Akt signaling axis in lung cancer

doi: 10.18632/aging.204826

Figure Lengend Snippet: Suppressing PRMT5 reduces the expression and stability of HIF-1α induced by hypoxia. ( A ) The mRNA expression of HIF-1α and PRMT5 was measured by qRT-PCR in PRMT5 depletion cells (HUVECs, n=4). *P < 0.05 vs. Scr. N.S. means not significant. ( B ) The HUVECs were infected with lentivirus containing scramble-shRNA or PRMT5-shRNA2, followed by CoCl 2 (200μM) treatment for 24h. The PRMT5 and HIF-α expression levels were determined by Western blotting (n=3). ( C ) The HUVECs were treated with vehicle or GSK591 (1μM) for five days and then were treated with CoCl 2 (200μM) for 24h. The HIF-1α expression level was determined by Western blotting (n=3). ( D ) The effect of different doses of GSK591 on the HIF-1α expression level induced by CoCl 2 as evaluated by Western blotting (n=3). ( E ) The HIF-1α stability was detected by Western blotting in PRMT5 depletion HUVECs upon cycloheximide (CHX, 20μg/mL) treatment at the indicated time points (n=3). ( F ) The HIF-1α stability was detected by Western blotting in GSK591-treated HUVECs upon cycloheximide treatment at the indicated time points (n=3). ( G ) The HUVECs were incubated with or without GSK591 (10μM) or infected with lentivirus containing PRMT5-shRNA2 and then pretreated with MG132 (10μM) and BAF-A1 (100nM) for 30 min before treating CoCl 2 . The HIF-1α expression levels were evaluated by Western blotting (n=3). ( H ) The HUVECs were infected with lentivirus containing scramble-shRNA or PRMT5-shRNA2 and then were transfected with vector or Flag-PRMT5 before treating CoCl 2 . The HIF-1α expression levels were evaluated by Western blotting (n=3).

Article Snippet: The lysosome inhibitor bafilomycin A1 (BAF-A1, cat# 1334) was purchased from TOCRIS.

Techniques: Expressing, Quantitative RT-PCR, Infection, shRNA, Western Blot, Incubation, Transfection, Plasmid Preparation