Journal: iScience

Article Title: Centrosomal P4.1-associated protein is a novel regulator of ESCRT pathway function during endosome maturation

doi: 10.1016/j.isci.2026.114659

Figure Lengend Snippet: CPAP depletion disrupts Rab5-to-Rab7 conversion (A) HeLa cells expressing control or CPAP shRNA were treated with untagged EGF for 60 min, stained for Rab5 and Rab7, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (B) HeLa cells expressing control or CPAP shRNA were transfected with GFP-Rab5 and mCherry-Rab7 constructs, treated with untagged EGF for 60 min, and imaged by Airyscan super-resolution microscopy. Left: maximum intensity-projection images of Z stacks; middle: single Z-plane of images; right: co-localization (yellow) was quantified by determining the percentages of GFP-Rab5-positive (green) puncta containing mCherry-Rab7 (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. The object-based co-localization macro tool FIJI was employed. (C) HeLa cells stably expressing control or CPAP-shRNA were transfected with control vector (GFP), GFP-Rab7 (WT; wild-type) or GFP-Rab7 (DN; dominant negative) vector constructs, treated with AF555-EGF ligand for 60 min, and stained for CD63 to mark MVBs/late endosomes, and imaged by confocal microscopy to determine AF555-EGF and CD63 co-localization. Left top row: GFP expression in control and Rab7 construct-expressing cells; left bottom rows: representative single Z-plane of images showing ligand-bound EGFR-CD63 co-localization; right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: The vector constructs expressing GFP-Rab5, GFP-Rab11, and mCherry-Rab7, GFP-Rab7-WT and GFP-Rab7-DN were purchased from Addgene. siRNA against HRS, ALIX and TSG101 were purchased from Santa Cruz Biotech.

Techniques: Expressing, Control, shRNA, Staining, Super-Resolution Microscopy, Transfection, Construct, Stable Transfection, Plasmid Preparation, Dominant Negative Mutation, Confocal Microscopy, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Myristoylated methionine sulfoxide reductase A is a late endosomal protein

doi: 10.1074/jbc.RA117.000473

Figure Lengend Snippet: Myristoylated MSRA is anchored to the membrane of late endosomes. A and C, COS7 cells were transiently transfected with three constructs, Stard3-FLAG, mRFP-Rab7, a late endosomal protein, and either (A) myristoylated MsrA-gfp or (C) nonmyristoylated MsrA-gfp. After 8 h, cells were treated with 20 mm NH4Cl for 16 h to alkalinize and enlarge late endosomes and lysosomes. The images within the white box of the upper panels are magnified in the lower panels. Scale bar, 10 μm. B and D, the merged images were scanned along the white arrow for relative fluorescence intensity with ZEN 2 software. The black arrows indicate the direction of scanning.

Article Snippet: The Met-307 and M307R/N311D mutants of the START domain were generated with the QuikChange kit. pDsRed2-Mito was purchased from Clontech (number 6975-1). mRFP-RAB7 was a gift from Ari Helenius (Addgene plasmid number 14436).

Techniques: Transfection, Construct, Fluorescence, Software

Journal: The Journal of Biological Chemistry

Article Title: Myristoylated methionine sulfoxide reductase A is a late endosomal protein

doi: 10.1074/jbc.RA117.000473

Figure Lengend Snippet: Subcellular fractionation demonstrates that myristoylated MSRA is enriched in the late endosome fraction. A, myristoylated MSRA is most prominent in fraction 1 from wildtype (WT) mouse liver and is not present in fractions prepared from the MSRA knockout liver (KO). Fraction 1 also contains most of the intact late endosomes. Each fraction was evaluated by immunoblot with these marker proteins: RAB7 (late endosome), mtHSP70 (mitochondria), myristoylated MSRA, total MSRA, and STARD3. The asterisk (*) indicates a nonspecific band. The molecular size markers of protein are indicated in kDa. The specificity of the mAb for the myristoylated form of MSRA is shown in panel B. 100 ng of purified, recombinant human myristoylated or nonmyristoylated MSRA were subjected to SDS-PAGE followed by immunoblotting by either the anti-myristoylated MSRA antibody or the general anti-MSRA antibody. As indicated under “Experimental procedures,” the concentration of protein was determined spectrophotometrically. However, to assure that equal amounts of the two proteins were loaded for immunoblotting, we also subjected 1 μg of each protein SDS-PAGE followed by staining with Coomassie Brilliant Blue. C, the anti-myristoylated MSRA and general anti-MSRA antibodies are specific for MSRA. This was demonstrated by immunoblotting homogenates of liver and kidney from WT and MSRA knockout mice. The asterisk (*) marks nonspecific bands detected by the general anti-MSRA antibody. Tubulin served as a loading control.

Article Snippet: The Met-307 and M307R/N311D mutants of the START domain were generated with the QuikChange kit. pDsRed2-Mito was purchased from Clontech (number 6975-1). mRFP-RAB7 was a gift from Ari Helenius (Addgene plasmid number 14436).

Techniques: Fractionation, Knock-Out, Western Blot, Marker, Purification, Recombinant, SDS Page, Concentration Assay, Staining

Journal: Microbiology Spectrum

Article Title: Cholesterol-rich lipid rafts mediate endocytosis as a common pathway for respiratory syncytial virus entry into different host cells

doi: 10.1128/spectrum.01192-25

Figure Lengend Snippet: RSV entry into cells triggers actin polymerization and endosome formation by cholesterol-rich lipid rafts. ( A ) RSV enters cells by actin-mediated endocytosis. HEp-2 or A549 cells were infected with RSV A2 (MOI = 30) (90 min, 37°C) and fixed (4% PFA, 15 min, room temperature). Cell nuclei were labeled with DAPI (cyan), lipid rafts with Alexa 647-CTB (blue), cholesterol with NBD-cholesterol (green), and F-actin with CellMask orange actin tracking dye (yellow), and images were acquired using confocal microscopy. Scale bar = 10 µm. Intensity profiles show colocalization along the direction of the yellow line. Pearson’s correlation between lipid rafts, cholesterol or F-actin, and RSV. n = 16 cells per group. Error bars represent a 95% confidence interval. ( B ) RSV trafficking from early to late endosomes. HEp-2 or A549 cells were infected with RSV A2 (MOI = 30) (150 min, 37°C) and fixed. Cell nuclei were labeled with DAPI (cyan), lipid rafts with Alexa 647-CTB (blue), and cholesterol with NBD-cholesterol (green). Immunodetection of RSV F (blue), RSV-FITC (green), EEA1, Rab7, and STX 6 (red) was performed using confocal microscopy. Scale bar = 10 µm. Intensity profiles show colocalization along the direction of the yellow line. Pearson’s correlation between lipid rafts, cholesterol or F-actin, and RSV. n = 16 cells per group. Error bars represent a 95% confidence interval.

Article Snippet: The Flotillin-1 (D2V7J) rabbit mAb (#18,934T), caveolin-1 (D46G3) rabbit mAb (#3267), EEA1 (C45B10) rabbit mAb (#3288), Rab7 (D95F2) rabbit mAb (#9367), Rab7 (E9O7E) mouse mAb (#95746), and syntaxin 6 (C34B2) rabbit mAb (#2869) were purchased from Cell Signaling Technology.

Techniques: Infection, Labeling, Confocal Microscopy, Immunodetection

Journal: Autophagy

Article Title: The activation of KSHV lytic cycle blocks autophagy in PEL cells

doi: 10.1080/15548627.2015.1091911

Figure Lengend Snippet: The autophagic flux was blocked in BC3 cells undergoing KSHV lytic cycle activation. ( A ) Analysis of K-bZIP lytic antigen, RAB7 and LC3-I/-II expression in the presence or in the absence of Baf (used for the last 3 h at 20 nM) in BC3 cells induced to enter the lytic cycle by T/B treatment for 36 h or ( B ) in the KSHV-negative BJAB cells undergoing the same treatment. ( C ) Kinetic analysis of the expression of K-bZIP, RAB7 and LC3-II and effects of Baf on the expression of K-bZIP, RAB7 and LC3-II in BC3 cells induced to enter the lytic cycle by T/B at the indicated time points. ACTB was used as loading control and a representative experiment out of 3 is shown. The histograms represent the mean plus SD of the densitometric analysis of the ratio of specific proteins to ACTB of 3 different experiments. ( D ) BC3 cells were transfected with pEGFP-LC3 plasmid and induced to enter the lytic cycle by T/B treatment for 0, 18 and 36 h. Lysosomes were stained in red with LysoTracker while autophagosomes were indicated by GFP-LC3 puncta. Bar: 5 micron. ( E ) Gallery of BC3 cells transfected with pDest-mCherry-EGFP-LC3B plasmid and treated with T/B for 36 h or starved for 2 h. The yellow puncta indicate autophagosomes while red puncta indicate autolysosomes. A representative experiment out of 3 is shown. Bar: 5 micron.

Article Snippet: The knockdown of BECN1 (Santa Cruz Biotechnology, sc-29797) or RAB7 (Santa Cruz Biotechnology, sc-29460) was performed in PEL cell lines using specific small interfering RNA duplex.

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Staining

Journal: Autophagy

Article Title: The activation of KSHV lytic cycle blocks autophagy in PEL cells

doi: 10.1080/15548627.2015.1091911

Figure Lengend Snippet: The autophagic flux was blocked in TRExBCBL1-Rta cells undergoing KSHV lytic cycle activation by doxycycline treatment. ( A ) Evaluation of the autophagic flux based on LC3-II accumulation in the presence or in the absence of Baf (used for the last 3 h at 20 nM) in TRExBCBL1-Rta cells induced to enter the lytic cycle by treatment with doxycycline for 48 h. TRExBCBL1-vector cells were used as control. ( B ) The expression of KSHV lytic antigen K8.1 and RAB7 was analyzed by western blot. TUBA1A was used as loading control and a representative experiment out of 3 is shown. The histograms represent the mean plus SD of the densitometric analysis of the ratio of LC3-II, K8.1 or RAB7 to TUBA1A of 3 different experiments. ( C ) Percentage of cells expressing K8.1 (red) lytic antigen analyzed by immunofluorescence. Nuclei were stained by DAPI (blue).

Article Snippet: The knockdown of BECN1 (Santa Cruz Biotechnology, sc-29797) or RAB7 (Santa Cruz Biotechnology, sc-29460) was performed in PEL cell lines using specific small interfering RNA duplex.

Techniques: Activation Assay, Plasmid Preparation, Expressing, Western Blot, Immunofluorescence, Staining

Journal: Autophagy

Article Title: The activation of KSHV lytic cycle blocks autophagy in PEL cells

doi: 10.1080/15548627.2015.1091911

Figure Lengend Snippet: RAB7 knockdown leads to an autophagic block in PEL cells. BCBL1 cells were knocked down for RAB7 or scramble (SC) treated and ( A ) RAB7 and ( B ) LC3-II expression level was evaluated in the presence or in the absence of Baf. TUBA1A was used as loading control and a representative experiment out of 3 is shown. The histograms represent the mean plus SD of the densitometric analysis of the ratio of LC3-II to TUBA1A of 3 different experiments.

Article Snippet: The knockdown of BECN1 (Santa Cruz Biotechnology, sc-29797) or RAB7 (Santa Cruz Biotechnology, sc-29460) was performed in PEL cell lines using specific small interfering RNA duplex.

Techniques: Blocking Assay, Expressing