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 Rab7 recruitment to the endosomes and ligand-bound, EGFR-positive endosomes HeLa cells expressing control or CPAP shRNA were treated with AF555-EGF ligand for the indicated time points, stained, and subjected to 4-color imaging by confocal microscopy. Images representing the detection of Rab7 and EEA1 (A), Rab7 and CD63 (B), and Rab7 (along with AF555-EGF) (C) are shown. Left (A–C): maximum intensity-projection images of confocal Z stacks. Middle (A and B) and right (C): single Z-plane of images. Right (A): co-localization (yellow) was quantified by determining the percentages of EEA1-positive (red) puncta containing Rab7 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells from 60 min time point of one representative experiment. Right (B): co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing Rab7 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells at 60 min time point of one representative experiment. The object-based co-localization macro tool FIJI was employed. Bottom left graph in (C): co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing Rab7 (green) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. Bottom right graph in (C): relative integrated fluorescence intensity values of Rab7 staining quantified 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: ∗<0.05, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test. Note: (A) and (C) present data from the same experiments where 4-color imaging was done and convey information on two different aspects based on three markers at a time. Since the visuals of the same cell can help with more reliable interpretation of the data, images of the same cell were used, where possible, for (A) and (C) with the same or different pseudo-color. Hence, duplication of some sub-images among (A) and (C) is intentional.

Article Snippet: Rab7 antibody , Santa Cruz , sc-376362.

Techniques: Expressing, Control, shRNA, Staining, Imaging, Confocal Microscopy, Fluorescence, MANN-WHITNEY

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: Rab7 antibody , Santa Cruz , sc-376362.

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

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: Rab5-to-Rab7 conversion and EGFR trafficking to late endosomes are restored in CPAP-depleted cells upon HRS, but not TSG101, overexpression (A) Schematic of the experimental strategy using control and CPAP-specific siRNA-treated HeLa cells with and without GFP-HRS or GFP-TSG101 expression. (B and C) Control and CPAP-specific siRNA-treated HeLa cells were subjected to mock or GFP-HRS or GFP-TSG101 vector transfection for 24 h, treated with untagged EGF for 60 min, and stained for Rab5 and Rab7 (B) or treated with AF555-EGF for 30 min and stained for Rab7 (C) and imaged by Lightning super resolution microscopy. Left: representative single Z-plane of images showing localization of Rab7 on Rab5-positive puncta (B) and AF555-EGF on Rab7-positive puncta (C) in cells with and without GFP-HRS or GFP-TSG101 expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in (B) and EGF-positive (red) puncta containing Rab7 (green, pseudo-color) in (C) 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: ∗<0.05, ∗∗ <0.01, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab7 antibody , Santa Cruz , sc-376362.

Techniques: Over Expression, Control, Expressing, Plasmid Preparation, Transfection, Staining, Super-Resolution Microscopy, MANN-WHITNEY

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: Rab5-to-Rab7 conversion and EGFR trafficking to MVB are restored in CPAP- and HRS-depleted cells upon exogenous CPAP expression (A) Schematic of the experimental strategy using control and CPAP- and HRS-specific siRNA-treated HeLa cells with and without siRNA-resistant GFP-CPAP expression. (B) Airyscan super-resolution microscopy images showing cells stained for Rab5 and Rab7 at 60 min time point. Left: representative single Z-plane of images showing localization of Rab5- and Rab7-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of Rab5-positive (green) puncta containing Rab7-positive (red) puncta in representative single Z-planes of each cell and quantified from multiple cells across at least three experiments. (C) Confocal microscopy images showing cells stained for CD63 and AF555-EGF at 60 min time point. Left: representative single Z-plane of images showing localization of CD63 − and EGF-positive puncta in cells with and without GFP expression. Right: co-localization (yellow) was quantified by determining the percentages of EGF-positive (red) puncta containing CD63-positive (green) 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 for (B) and (C). Zoomed images correspond to the dashed inset boxes of the indicated images. Scale bars: 10 μm. p values: ∗∗∗<0.001, ∗∗∗∗<0.0001 by unpaired nonparametric Mann-Whitney test.

Article Snippet: Rab7 antibody , Santa Cruz , sc-376362.

Techniques: Expressing, Control, Super-Resolution Microscopy, Staining, Confocal Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

doi: 10.1101/2025.11.03.686439

Figure Lengend Snippet: Fluorescence imaging showing fraction of CTR1 (green) localized with respective endocytic vesicles (red) while DAPI (blue) marks the nucleus at copper (50 µM) and CDDP (25µM & 50µM). The Scale bar represents 10 microns. (A) Immunofluorescence image showing fraction of Ctr1 (green) with early endosomal marker EEA1 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-EEA1. (B) Immunofluorescence image showing fraction of Ctr1 (green) with retromer controlled Recycling endosomal vesicle VPS35 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-VPS35. (C) Immunofluorescence image showing fraction of Ctr1 (green) with late endosomal marker Rab7 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab7. (D) Immunofluorescence image showing fraction of Ctr1 (green) with retromer late endosomal marker Rab9 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab9. (E) Immunofluorescence image showing fraction of Ctr1 (green) with lysosomal resident protein Lamp2 (Red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Lamp2. (F) Quantitative analysis showing fraction of CTR1 residing in different endocytic vesicles upon 1 hr. treatment of copper (100 µM) and CDDP (100 µM) respectively graphically represented using a Box and Whisker plot with jitter points (n>50). The box represents the 25 to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5 × interquartile range (IQR) from the first and third quartile. ∗ p < 0.05, ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: The primary antibodies utilized in immunofluorescence studies are as follows: Rabbit anti-FLAG M2 ( :600,CST #14793), Rabbit Ctr1(1:800,# ab129067 Abcam), Mouse Na/K ATPase ( :600,#MA3-929 Invitrogen), Mouse VPS35 ( :500,#sc-374372, Santa Cruz Biotechnology), Mouse Rab7 ( :50,#sc-376362, Santa Cruz Biotechnology), Mouse Lamp2 ( :150, #H4B4 DSHB), Mouse Rab9 ( :100, #MA3-06 Invitrogen), Mouse EEA1(1:100 #610457, 1:400; BD Biosciences).The Secondary antibodies utilized in immunofluorescence studies are as follows: Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 ( :2000, #A-21206 Invitrogen), Donkey anti-Mouse Alexa 555 (#A-32773, 1:2000; Invitrogen).

Techniques: Fluorescence, Imaging, Immunofluorescence, Marker, Whisker Assay

Journal: bioRxiv

Article Title: Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

doi: 10.1101/2025.11.03.686439

Figure Lengend Snippet: (A) Immunofluorescence image showing fraction of Ctr1 (green) with early endosomal marker EEA1 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-EEA1. (B) Immunofluorescence image showing fraction of Ctr1 (green) with retromer controlled Recycling endosomal vesicle VPS35 (red) in HEK293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-VPS35. (C) Immunofluorescence image showing fraction of Ctr1 (green) with late endosomal marker Rab7 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab7. (D) Immunofluorescence image showing fraction of Ctr1 (green) with retromer late endosomal marker Rab9 (red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Rab9. (E) Immunofluorescence image showing fraction of Ctr1 (green) with lysosomal resident protein Lamp2 (Red) in Hek293T cell line. The marked area on the ‘merge’ panel is enlarged in ‘inset’. The arrowhead shows colocalization between Ctr1-Lamp2. (F) Quantitative analysis showing fraction of CTR1 residing in different endocytic vesicles upon 1 hr. treatment of copper (100 µM) and CDDP (100 µM) respectively graphically represented using a Box and Whisker plot with jitter points (n>50). The box represents the 25 to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5 × interquartile range (IQR) from the first and third quartile. ∗ p < 0.05, ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: The primary antibodies utilized in immunofluorescence studies are as follows: Rabbit anti-FLAG M2 ( :600,CST #14793), Rabbit Ctr1(1:800,# ab129067 Abcam), Mouse Na/K ATPase ( :600,#MA3-929 Invitrogen), Mouse VPS35 ( :500,#sc-374372, Santa Cruz Biotechnology), Mouse Rab7 ( :50,#sc-376362, Santa Cruz Biotechnology), Mouse Lamp2 ( :150, #H4B4 DSHB), Mouse Rab9 ( :100, #MA3-06 Invitrogen), Mouse EEA1(1:100 #610457, 1:400; BD Biosciences).The Secondary antibodies utilized in immunofluorescence studies are as follows: Donkey anti-Rabbit IgG (H + L) Alexa Fluor 488 ( :2000, #A-21206 Invitrogen), Donkey anti-Mouse Alexa 555 (#A-32773, 1:2000; Invitrogen).

Techniques: Immunofluorescence, Marker, Whisker Assay

Journal: mBio

Article Title: Herpes simplex virus diverts CIN85 endosomal cargo for exocytosis to evade antiviral responses: a novel role for the viral immediate-early protein ICP0

doi: 10.1128/mbio.02143-25

Figure Lengend Snippet: ICP0 localizes on the surface of the CIN85 structures, along with early and late endosome, autophagy, and innate immunity factors. ( A ) Vero cells were transfected with a CIN85-Flag expressing plasmid for 24 h followed by HSV-1(F) infection (10 PFU/cell). Cells were fixed at 14 h post-infection and stained with an ICP0 and a Flag antibody. Images were captured using a CSU-W1 SoRa confocal microscope. ( B and C ) Vero cells were transfected with plasmids expressing CIN85-Flag, mCherry-Rab5, mRFP-Rab7, mCherry-LC3, mCherry-p62, mCherry-ATG5, YFP-Sp100A, and CD63-EGFP or co-transfected with a CIN85-Flag-expressing plasmid and plasmids expressing the above-mentioned proteins. At 24 h post-transfection, the cells were infected with HSV-1(F) (10 PFU/cell). Cells were fixed at 14 h post-infection and probed with an ICP0 or a Flag antibody. Images were captured using a TCS SP8 STED microscope.

Article Snippet: Rab7 , Mouse , 1:1,000 , Santa Cruz , sc-376362.

Techniques: Transfection, Expressing, Plasmid Preparation, Infection, Staining, Microscopy

Journal: mBio

Article Title: Herpes simplex virus diverts CIN85 endosomal cargo for exocytosis to evade antiviral responses: a novel role for the viral immediate-early protein ICP0

doi: 10.1128/mbio.02143-25

Figure Lengend Snippet: HSV-1(F) infection induces exocytosis of CIN85 and of components colocalizing with CIN85. ( A and Β ) hTERT-HEL cells were uninfected, infected with the WT virus or various ICP0 mutants (0.5 PFU/cell). The cells were harvested at 48 h post-infection, and total EVs were analyzed for the indicated proteins. Equal amounts of proteins from total cell lysates were included as controls. ( C and D ) U2OS cells were infected with HSV-1(F), ΔICP0, and ICP0 delta244–277 virus (0.5 PFU/cell) or remained uninfected. EVs were collected at 48 h post-infection, and equal amounts were analyzed for CIN85 exocytosis. Equal amounts of proteins from total cell lysates were analyzed for CIN85, VP16 which served as a control for the infection, and β-actin which served as a loading control. ( Ε and F ) hTERT-HEL cells were infected with HSV-1(F), ΔICP0, and ICP0 delta244–277 virus (0.5 PFU/cell) or remained uninfected. EVs were collected from culture supernatants at 48 h post-infection and analyzed for Rab5, Rab7, ATG5, Sp100, p62/SQSTM1, Alix, and LC3-B. Equal amounts of proteins from total cell lysates were analyzed for the same proteins. ICP0 or VP16 was used as controls for the infection and β-actin as a loading control.

Article Snippet: Rab7 , Mouse , 1:1,000 , Santa Cruz , sc-376362.

Techniques: Infection, Virus, Control