Journal: Frontiers in Molecular Biosciences

Article Title: Loss of ARF5 impairs recovery after lysosomal damage

doi: 10.3389/fmolb.2025.1699266

Figure Lengend Snippet: Lipid transfer proteins OSBP and ORP9 are recruited to damaged lysosomes in an ARF-independent manner. (A) HeLa cells were transfected with empty vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 35 min, fixed and stained for endogenous OSBP (green), LAMP1 (red), and TGN46 (magenta), and imaged with confocal microscopy. Scale bars indicate 10 µm. (B) HEK293 cells stably expressing tagged TMEM192 were transfected with empty pLKO vector (shCTRL) or pLKO containing ARF1 or ARF5 shRNAs. Untreated and LLOME (1 mM)-treated cells were subjected to the LysoIP protocol. Precipitated lysosomes were immunoblotted for endogenous OSBP. (C) HeLa cells were transfected with empty vector (shCTRL), ARF5, or ARF6 shRNAs. Cells were treated or not with LLOME (0.5 mM) for 20 min, fixed and stained for ORP9 (green), LAMP1 (red), and F-actin (blue), and imaged with confocal microscopy. Scale bars indicate 10 µm. (D) Quantification of data in (C) n = 28 cells. Error bars represent mean +SD. Data were analyzed with two-way ANOVA with Dunnett’s multiple comparisons test with a single pooled variance. *p < 0.05.

Article Snippet: The antibodies we used were: rabbit antibody to LAMP1 (D2D11) (Cell Signaling Technology, Inc.), Cat No. 9091S, IF (1:400) WB (1:1200); mouse antibody to LAMP1 (1D4B) from Developmental Studies Hybridoma Bank; mouse antibody to GFP (Proteintech) Cat No.:66002-1-Ig WB (1:100,000); mouse antibody to HA (16B12) (Biolegend) WB (1:4000); rabbit antibody to mCherry (Sigma-Aldrich) Cat No.SAB2702295-100UL WB (1:10,000); rabbit antibody to OSBP (Sigma) Cat no. HPA039227 WB (1:1100) IF (1:100); rabbit antibody to ORP9 from Dr. Neale Ridgway, Dalhousie University, IF (1:1000); mouse antibody to Golgin97 (Molecular probes) Cat No. CDF4 A-21270 WB (1:500); rabbit antibody to ARF5 (Novus Biologicals) Cat No. NBP1-31005 WB (1:2500); sheep antibody to TGN46 (Serotec, Oxford United Kingdom); mouse antibody to Gal-3 (B-2) (Santa Cruz Biotechnology, Inc.) Cat No. sc-25279 IF (1:100); IRDye 800CW donkey anti-mouse secondary antibody (Li-COR) 926-32212 WB (1:10,000); IRDye 680RD goat anti-rabbit secondary antibody (Li-COR) 926-68071 WB (1:10,000); Alexa Fluor 488 donkey anti-mouse (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 488 donkey anti-rabbit (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 568 donkey anti-mouse (ThermoFisher Scientific (Rockford, IL) IF (1:100); Alexa Fluor 568 donkey anti-rabbit (ThermoFisher Scientific (Rockford, IL) IF (1:100).

Techniques: Transfection, Plasmid Preparation, Staining, Confocal Microscopy, Stable Transfection, Expressing

Journal: Science Advances

Article Title: The nuclear import receptor importin-7 targets HPV from the Golgi to the nucleus to promote infection

doi: 10.1126/sciadv.adz6792

Figure Lengend Snippet: ( A ) HeLa cells were mechanically homogenized and fractionated through a 0.5 to 1.6 M sucrose gradient. Portions of each fraction (#1 to #11, top to bottom) were immunoblotted with the indicated antibodies to identify organelle markers. Fractions #3 containing TGN46 and GM130 represents the Golgi-enriched fraction. ( B ) Fraction #3 collected from (A) was subjected to IP with an anti-GM130 antibody or a control IgG and then immunoblotted with the indicated antibodies. ( C ) HeLa S3 cells were transfected with 10 nM Scr or IPO7 siRNA #2 for 48 hours and then uninfected or infected with HPV16.L2F (MOI, ~100). PLA (green) for IPO7 and GM130 was performed at 30 hpi; nuclei were stained with DAPI (blue). Similar results were obtained in two independent experiments. Scale bar, 10 μm. ( D ) PLA fluorescence intensity per cell (>80 cells) as in (C), shown as individual values, means, and SDs, with statistical significance determined by one-way ANOVA. *** P < 0.001 and **** P < 0.0001. ( E ) Uninfected HeLa cells were transfected with the mNG2-SREBP1 reporter construct for 48 hours and then with 10 nM Scr or IPO7 siRNA #2 for another 48 hours. Samples were fixed and permeabilized, and nuclei were stained with DAPI. The nuclear import of mNG2-SREBP1 was indicated by colocalization of mNG2-SREBP1 fluorescence (green) and DAPI (blue). Similar results were obtained in two independent experiments. Scale bar, 10 μm. ( F ) Ratio of nuclear to total mNG2-SREBP1 intensity per cell (>26 mNG-positive cells) as in (E) is plotted as individual values, means, and SDs, with statistical significance determined by two-tailed, unequal variance t test. **** P < 0.0001.

Article Snippet: TGN46 , Rabbit , 13573-1-AP; Proteintech , WB.

Techniques: Control, Transfection, Infection, Staining, Fluorescence, Construct, Two Tailed Test