Journal: bioRxiv

Article Title: CLIC-dependent internalization of caveolin-1 to lysosomal vacuoles in response to osmotic regulation

doi: 10.1101/2025.08.26.672461

Figure Lengend Snippet: A) MDA-MB-231 cells were incubated in isotonic media, in hypotonic media for 5, 15, 30 and 60 minutes or under Recovery conditions in which hypotonic media was replaced after 60 minutes with isotonic media for 5, 15, 30 or 60 minutes. Cells were labeled for Cav1, fixed and imaged by TIRF widefield imaging. Average Cav1 intensity per cell was quantified and normalized to isotonic control. Representative images of select conditions are shown. (n=3 independent experiments with >30 cells for each condition; ANOVA with Dunnett’s post-test comparing to isotonic control; **p < 0.01; ***p < 0.001; Scale bar: 2µm). B) STED imaging of anti-Cav1 labeling MDA-MB-231 cells incubated in isotonic media or in hypotonic media for 15 or 60 minutes. Cav1 endocytic vacuoles were counted per cell. (n>28 from three independent experiments for isotonic and hypotonic 15 minutes, four independent experiments for other conditions; (ANOVA with Dunnett post-test comparing to isotonic control; *p < 0.05; ****p < 0.0001; Scale bar: 4µm; Inset scale bar: 1µm). C) MDA-MB-231 cells incubated with isotonic or hypotonic media for 60 minutes were fixed and labeled for Cav1, CD44 and β1-integrin (Scale bar: 4µm). The diameter of individual Cav1, CD44, and β1-integrin positive endosomes was measured from three independent experiments.

Article Snippet: Primary antibodies: rabbit anti-Cav1 mAb (3267) was purchased from Cell Signaling Technology, rat anti-CD44 mAb (22530) from Novus, and mouse anti-β1 integrin mAb (sc-53711) from Santa Cruz.

Techniques: Incubation, Labeling, Imaging, Control

Journal: bioRxiv

Article Title: CLIC-dependent internalization of caveolin-1 to lysosomal vacuoles in response to osmotic regulation

doi: 10.1101/2025.08.26.672461

Figure Lengend Snippet: A) MDA-MB-231 cells were incubated with anti-CD44 and anti-β1 integrin antibodies in isotonic media for 60 minutes at 4°C or 37°C or in hypotonic media for 5, 15, 30 or 60 minutes at 37°C. As indicated, an acid wash treatment was performed at 4°C to remove surface antibodies and cells were fixed and labeled with secondary antibodies to anti-CD44 and anti-β1 integrin as well as for endogenous Cav1. (n= >30 cells per condition from three independent experiments; ANOVA with Dunnett post-test comparing each condition to isotonic 4°C control; *p < 0.05; ***p < 0.001; ****p < 0.0001; Scale bars: 2 µm). B) MDA-MB-231 cells were transfected with wild-type CDC42-GFP or dominant negative (Dom Neg) CDC42-GFP, incubated with isotonic or hypotonic media for 60 minutes and then fixed and labeled for Cav1 and CD44. CDC42-GFP was also imaged and the number of Cav1 endocytic vacuoles counted per cell (n>30 cells from three independent experiments; ANOVA with Dunnett’s post-test comparing to isotonic control; ****p < 0.0001; Scale bar: 2µm).

Article Snippet: Primary antibodies: rabbit anti-Cav1 mAb (3267) was purchased from Cell Signaling Technology, rat anti-CD44 mAb (22530) from Novus, and mouse anti-β1 integrin mAb (sc-53711) from Santa Cruz.

Techniques: Incubation, Labeling, Control, Transfection, Dominant Negative Mutation

Journal: bioRxiv

Article Title: CLIC-dependent internalization of caveolin-1 to lysosomal vacuoles in response to osmotic regulation

doi: 10.1101/2025.08.26.672461

Figure Lengend Snippet: A) MDA-MB-231 cells were transfected with LAMP-GFP, incubated with isotonic or hypotonic media for 60 minutes and then fixed and labeled for Cav1. LAMP-GFP was also imaged and the number of Cav1 endocytic vacuoles counted per cell (n>30 from cells three independent experiments with >30 cells for each condition; ANOVA with Dunnett’s post-test comparing to isotonic control; ****p < 0.0001; Scale bar: 2 µm). B) Cav1 CRISPR knockout MDA-MB-231 cells were transfected with Cav1-WT-HA or Cav1-K*R-HA lysine mutant, incubated with isotonic or hypotonic media for 15 or 60 minutes and then fixed and labeled for Cav1 and CD44. (n>29 cells from three independent experiments; ANOVA with Tukey post-test comparing WT to K*R at the same isotonic or hypotonic conditions and the respective hypotonic condition to isotonic control; Scale bar: 2µm). C) MDA-MB-231 cells were transfected with LAMP1-mScarlet and Cav1-Halo, where indicated pre-treated with 100nM BafA for 2 hours, labeled with Halo-LIVE RED for 30 minutes and then incubated for 30 minutes with 1uM LysoSensor green and LIVE RED Halotag ligand. Cells were then placed in isotonic or hypotonic imaging media and multiple cells imaged over a period of 30 minutes in isotonic DMEM or after 30 minutes of hypotonic shock. LysoSensor and Cav1 intensity within LAMP1-positive lysosomes were quantified. (n>38 cells from three independent experiments; ANOVA with Tukey’s post-test; *p < 0.05; ****p < 0.0001; Scale bar: 2 µm).

Article Snippet: Primary antibodies: rabbit anti-Cav1 mAb (3267) was purchased from Cell Signaling Technology, rat anti-CD44 mAb (22530) from Novus, and mouse anti-β1 integrin mAb (sc-53711) from Santa Cruz.

Techniques: Transfection, Incubation, Labeling, Control, CRISPR, Knock-Out, Mutagenesis, Imaging

Journal: bioRxiv

Article Title: CLIC-dependent internalization of caveolin-1 to lysosomal vacuoles in response to osmotic regulation

doi: 10.1101/2025.08.26.672461

Figure Lengend Snippet: A) Cell volume of MDA-MB-231 and PC3 cells switched from isotonic to hypotonic media was measured by live phase imaging. Black line indicates addition of hypotonic media (n=3 independent experiments; each experiment includes 2-3 technical replicates and each technical replicate consists of 9 ROIs). B) MDA-MB-231 and PC3 cells were incubated in isotonic media, or hypotonic or hypertonic media for 5, 15, 30 and 60 minutes. Cells were labeled for Cav1 and CD44 and Cav1 endocytic vacuoles counted per cell. Representative images for select time points are shown. (n>28 cells from three independent experiments; ANOVA with Dunnett’s post-test comparing each condition to isotonic control; *p < 0.05; **p < 0.01; ****p < 0.0001; Scale bar: 2 µm). C) PC3 cells and PC3 cells stably transfected Cavin-1-GFP were incubated in isotonic, hypotonic and hypertonic shock media for 60 minutes and then fixed and labeled for Cav1. Cavin-1-GFP was imaged in the GFP channel and Cav1 endocytic vacuoles counted per cell. (n>28 cells from three independent experiments; ANOVA with Tukey’s post-test and comparing PC3 to PC3+Cavin-1 and each condition with the respective isotonic control; **p < 0.01; ***p < 0.001; ****p < 0.0001; Scale bar: 2 µm).

Article Snippet: Primary antibodies: rabbit anti-Cav1 mAb (3267) was purchased from Cell Signaling Technology, rat anti-CD44 mAb (22530) from Novus, and mouse anti-β1 integrin mAb (sc-53711) from Santa Cruz.

Techniques: Imaging, Incubation, Labeling, Control, Stable Transfection, Transfection

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Distribution of CD44 + cells within intestinal epithelium. a , b Immunohistochemical (IHC) staining for Lgr5 + ISCs ( black dotted lines ) in vivo. c , d IHC staining for CD44 + cells ( black dotted lines ) in vivo. a , c Magnification ×400. Bars 50 μm. b , d Magnification ×1000. Bars 20 μm. e–n Immunocytochemical (ICC) staining for CD44 + cells in vitro. e , j Differential interference contrast (DIC) imaging. f , k Propidium iodide (PI) staining for nuclei. g , l Fluorescein isothiocyanate (FITC) for CD44 + cells ( white arrowheads crypt cells strongly positive for CD44). h , m Overlay of PI image and FITC image. i , n Overlay of FITC image and DIC image. e–i Magnification ×200. Bars 200 μm. j–n Magnification ×630. Bars 100 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Immunohistochemical staining, Immunohistochemistry, In Vivo, Staining, In Vitro, Imaging

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Identification of CD44 + cells. a–a’’’’’ Fluorescence-activated cell sorting (FACS) analysis for cellular phenotype. a Isotype control, IgG2a-phycoerythrin (PE). a’ CD31-PE. a’’ CD34-PE. a’’’ Isotype control, IgG2b-allophycocyanin (APC). a’’’’ CD44-APC ( lo low-positive for CD44, hi high-positive for CD44). a’’’’’ CD45-APC. b Development of CD44 + ISC in 3D-culture system. Numbers represent days. Top Magnification ×400. Bar 50 μm. Bottom Magnification ×200. Bars 100 μm. c–c’’ Colony-forming efficacies of CD44 - cells and CD44 + cells. c CD44 - cells in 3D-culture system for 14 days. c’ CD44 + cells in 3D-culture system for 14 days. c , c’ Magnification ×40. Bars 500 μm. c’’ Comparision of colony-forming efficacy per 100 sorted cells seeded in one well of a 96-well plate. CD44 - group in 48 wells; CD44 + group in 48 wells. Data represent means ± SD of 48 independent measurements ( n = 48). Bars SD values. Paired t -test was used for data analysis. * P ≤ 0.05 represents high significance (CD44 + group versus CD44 - group). All experimental procedures were repeated twice. d–d’’’’ Transmission electron microscope imaging of CD44 + ISC differentiation. CD44 + ISC were cultured in the 3D-system for 6 days and formed a cystic structure. d Cystic structure of a single CD44 + ISC-derived organoid at 6 days. Boxed areas are shown at higher magnification in d’–d’’’’ ( Lu lumen). Magnification ×400. Bar 50 μm. d’ Absorptive cell ( Ab ). d’’ Endocrine cell ( En ). d’’’ Goblet cell ( Go ). d’’’’ Paneth cell ( Pa ). Black arrowhead in d’ indicates brush border. Black arrowheads in d’’–d’’’’ indicates granules. d’–d’’’’ Magnification ×1500. Bars 5 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Fluorescence, FACS, Control, Transmission Assay, Microscopy, Imaging, Cell Culture, Derivative Assay

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: CD44 + ISCs resemble CBC stem cells. a–a’’ Strategy for sorting CD44 - , CD44 low+ and CD44 hi+ subpopulations by using the FACS technique ( R1 determining the cell-zone, R2 determining viable cells, PI propidium iodide, APC allophycocyanin). b Semi-quantitative reverse transcription (RT) followed by the polymerase chain reaction (PCR) for ISC-related gene-expression in sorted cells. Fold expression values were normalized to the CD44 - group. Data represent means ± SD of six independent measurements ( n = 6). Bars indicate value of SD. The paired t -test was used for data analysis. * P ≤ 0.05 represents high significance (CD44 hi+ group versus CD44 low+ group); $ P ≤ 0.05 represents low significance (CD44 hi+ group versus CD44 low+ group); ns represents no statistic differences between the CD44 hi+ group and CD44 low+ group. P -values for Lgr5 , Bmi1 , Hopx , mTERT , Ascl2 , Smoc2 , Lrig1 , Rnf43 and Prominin-1 are respectively 0.016, 0.036, 0.010, 0.844, 0.007, 0.001, 0.041, 0.005 and 0.859. c , c’ Colony-forming efficacy of CD44 low+ and CD44 hi+ cells in 3D-culture system for 14 days. c CD44 low+ group. c’ CD44 hi+ group. Magnification ×40. Bars 500 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Expressing

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Epithelial homeostasis in CD44 + ISC-derived organoid. a , b TUNEL staining for apoptotic cells in normal epithelium. c , d IHC staining of Ki67 for proliferative cells within normal epithelium. a , c Magnification ×200. Bars 100 μm. b , d Magnification ×400. Bars 50 μm. e–j Epithelial homeostasis in vitro. e–g TUNEL staining for apoptotic cells in CD44 + ISC-derived organoid. e DAPI staining ( blue ) for nuclei. f dUTP-FITC ( green ) for apoptotic cells. g DAPI image merged with dUTP-FITC image. Magnification ×100. Bars 200 μm. h–j ICC staining for proliferative cells in CD44 + ISC-derived organoid. h DAPI staining for nuclei. i Ki67-FITC for proliferative cells. j DAPI image merged with Ki67-FITC image. Magnification ×200. Bar 100 μm. k Representation of an organoid

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Derivative Assay, TUNEL Assay, Staining, Immunohistochemistry, In Vitro