Journal: bioRxiv

Article Title: Plasmodium falciparum exploits CD44 as a co-receptor for erythrocyte invasion

doi: 10.1101/2023.04.12.536503

Figure Lengend Snippet: (A) Flow cytometry-based assay to quantify binding between free P. falciparum merozoites and recombinant CD44-Fc or CD99-Fc proteins. Free merozoites were incubated with recombinant proteins, and binding was detected using anti-CD44 or anti-CD99 antibodies. (B) Flow cytometry-based assay to quantify binding between free P. falciparum merozoites and recombinant CD44-His or GYPC-His proteins. Histograms depicting merozoite fluorescence upon incubation with recombinant His-tagged proteins and detection with anti-His antibody. (C) Images of immunofluorescence assays of free P. falciparum merozoites after binding to rCD44-His, and its co-localization with the merozoite protein RAP1. Images were taken with a Keyence BZ-X700 all-in-one fluorescence microscope at 100x magnification under oil immersion.

Article Snippet: Protein A Dynabeads (1.5 mg) were incubated with 8 µg rCD44-Fc (R&D systems 3660-CD), rCD44-Fc (SinoBiological 12211-H02H), rCD55-Fc (SinoBiological 10101-H02H), IgG 1 -Fc (R&D systems 110-HG), or no protein in 200 µl antibody binding and washing buffer (Invitrogen kit 10006D) for one hour at RT.

Techniques: Flow Cytometry, Binding Assay, Recombinant, Incubation, Fluorescence, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Plasmodium falciparum exploits CD44 as a co-receptor for erythrocyte invasion

doi: 10.1101/2023.04.12.536503

Figure Lengend Snippet: (A) Mass spectrometry results from affinity purification experiments in which bead-immobilized rCD44-Fc or rFc were incubated with lysate from P. falciparum strain 3D7 schizont-stage parasites. Heat map shows the normalized spectral counts (based on protein size) of the most abundant P. falciparum proteins detected and their enrichment in rCD44-Fc compared to rFc. NaN indicates no counts. The table shows Log probability, raw number of spectra and unique peptides, and coverage of EBA-140 and EBA-175 proteins found in rCD44-Fc lane. (B) Western blot of independent affinity purification experiment in which bead-immobilized rCD44-Fc or rFc were incubated with P. falciparum schizont-stage lysate, followed by immunoblotting for EBA-175 or EBA-140. SN, supernatant. (C) Flow cytometry-based binding assays in which recombinant region RII of EBA-175-His (left) or EBA-140-His (right) were incubated with beads coated with rCD44-Fc, rFc, or no protein. Binding was detected using an anti-His antibody and a fluorescent secondary antibody. (D) Flow cytometry-based binding assays between rCD55-Fc or rFc and region RII of EBA-175-His (left) or EBA-140-His (right). (E) Binding assays of RII EBA-175 (left) or RII EBA-140 (right) with rCD44-Fc in the presence of anti-CD44 monoclonal antibody BRIC 222 or isotype control BRIC 170.

Article Snippet: Protein A Dynabeads (1.5 mg) were incubated with 8 µg rCD44-Fc (R&D systems 3660-CD), rCD44-Fc (SinoBiological 12211-H02H), rCD55-Fc (SinoBiological 10101-H02H), IgG 1 -Fc (R&D systems 110-HG), or no protein in 200 µl antibody binding and washing buffer (Invitrogen kit 10006D) for one hour at RT.

Techniques: Mass Spectrometry, Affinity Purification, Incubation, Western Blot, Flow Cytometry, Binding Assay, Recombinant, Protein Binding, Control

Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: Identification and distribution of CD44-positive cancer stem-like cells in human pNET tissues. A . Human pNET specimens in patient 1 were co-stained with CD44 and α-SMA antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (blue) by immunofluorescence microscopy. CD44-positive cancer cells (red) marked by yellow arrowheads were close to the α-SMA positive (green) blood vessels. B : Human pNET specimens in patient 2 were co-stained as in A . CD44-positive cancer cells (green) were indicated by yellow arrowheads, which were close to the α-SMA positive (red) blood vessels. Note: A group of CD44-positive cells appear to distribute within the α-SMA-positive vascular niche or nearby blood vessels. Shown are representative images from different individual patients. Bar = 10 or 20 µm. C . Human pNET tissues were co-stained with CD44 antibodies and CD45 antibodies followed by appropriate secondary antibodies. Overlay images were collected by immunofluorescence microscopy. CD44-positive (green, yellow arrowheads) and both CD44-positive and CD45-positive cells (orange, blue arrowheads) were observed under a fluorescence microscope. Images were acquired by a fluorescence microscope equipped with a CCD camera. Shown are representative images from two individual patients. Bar = 10 µm. Note: Cells positive with both CD44 and CD45 are smaller and regarded as lymphatic cells (orange), a subset of cancer cells are bigger and positive for CD44 but negative for CD45, indicating their stem-like phenotype.

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Staining, Immunofluorescence, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: Stem-like phenotype of cancer cells in pNETs. A . A human pancreatic tissue control (top panel) and human pNET specimens from two individual patients (middle and lower panels) were co-stained with CD44 and α-SMA antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (blue) by immunofluorescence microscopy. CD44-positive cancer stem-like cells were marked by yellow arrowheads, which were close to the α-SMA positive (green, red arrowheads) blood vessels. The fluorescence images were acquired by an immunofluorescence microscope equipped with a CCD camera, and representative images are shown, along with H & E staining for tumor tissue structures. Bar = 10 µm.

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Control, Staining, Immunofluorescence, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: PKD-1 signaling in the maintenance of cancer stem-like features in pNETs. A . Distribution of PKD-1 + and CD44 + CSCs within the vascular niche. Human pNET specimens were co-stained with CD44 and PKD-1 antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (Blue). Stem-like cells with CD44-positive (green), PKD-1-positive (red) or both positive (pink) were observed under a fluorescence microscope. A few CD44-positive cancer stem-like cells tended to accumulate near the vascular lumen (red arrow heads), and PKD-1-positive CSCs might be leaving tumor nests (stars) for the vascular lumen. The fluorescence images were acquired by an immunofluorescence microscope equipped with a CCD camera. Shown are representative images. Bar = 10 µm. B . BON and QGP-1 cells were transfected with siRNA control and siPKD-1 to knock down PKD-1. Knockdown efficiency was confirmed by Western Blots (upper panel). The control and BON cells with knocked-down PKD-1 were subjected to tumorsphere formation assays. Images were acquired by the OLYMPUS CK30 microscope. Representative images are shown for tumorsphere formation (lower panel). C . Cell lysates were extracted from BON and QGP-1 cells exposed to the vehicle control, 10 μ M LPA, 2 μ M CRT0066101, or their combinations after 24 hours. The expression levels of phosphorylated PKD-1 and PKD-1 were detected by Western blots. Shown are representative images. D . BON and QGP-1 cells were exposed to 10 μ M LPA, 2 μ M CRT0066101, or their combination for 24 hours, and total RNA was extracted for the detection of mRNA levels of genes related to stemness properties by RT-qPCR. E . Effect of PKD inhibitor in tumorsphere formation. BON cells were cultured in complete MammoCult(tm) medium with the treatment of 10 μ M LPA, 2 μ M CRT0066101, or their combination for 7 days. The mammary spheres were counted under the OLYMPUS CK30 microscope. F . Control and BON cells with PKD-1 knockdown were exposed to 10 μ M LPA, 2 μ M CRT0066101, or their combination for 24 hours, and total RNA was extracted for the detection of mRNA levels of genes related to stemness properties by RT-qPCR. Note: Triplicate experiments were performed, and the results are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Staining, Fluorescence, Microscopy, Immunofluorescence, Transfection, Control, Knockdown, Western Blot, Expressing, Quantitative RT-PCR, Cell Culture

Journal: bioRxiv

Article Title: PKD-1 Signaling Is Required for the Maintenance of CSCs with Epithelial-mesenchymal Plasticity in Pancreatic Neuroendocrine Tumors

doi: 10.1101/2022.02.17.480869

Figure Lengend Snippet: Distribution of CD44-and/or PKD-1-positive cancer stem-like cells in pNET tissues. Human pNET specimens were co-stained with CD44 antibodies and PKD-1 antibodies followed by appropriate secondary antibodies, with DAPI staining the nuclei (blue). CD44-positive (green), PKD-1-positive (red) or both positive were observed under a fluorescence microscope. Cancer cells with high levels of both CD44 and PKD-1 (yellow arrowheads) likely left the tumor nests (white stars) and accumulated in the nearby vascular lumen (red arrowheads). Fluorescence images were acquired by an immunofluorescence microscope equipped with a CCD camera. Shown are representative images from two individual patients. Bar = 10 µm. Note: These are additional pictures for .

Article Snippet: Antibodies for immunostaining included rabbit anti-human/mouse PKD-1 (SAB4502371) and mouse anti-human/mouse α-smooth muscle actin (α-SMA, A2547) (Sigma-Aldrich), mouse anti-human/rat CD44 (5640S) and HRP-linked anti-rabbit IgG (7074) (Cell Signaling Technology), CD45 monoclonal Antibody (14-9457-80), AlexaFluor 594 conjugated donkey anti-rabbit IgG (A21207), AlexaFluor 594 conjugated donkey anti-mouse IgG(A21203), AlexaFluor 594 conjugated donkey anti-goat IgG (A11058), AlexaFluor 488 conjugated donkey anti-goat IgG(A11055), AlexaFluor 488 conjugated donkey anti-rabbit IgG (A21206), and AlexaFluor 488 conjugated goat anti-mouse IgG (A11001) (Invitrogen).

Techniques: Staining, Fluorescence, Microscopy, Immunofluorescence

Journal: Biology of reproduction

Article Title: Expression of two isoforms of CD44 in human endometrium.

doi: 10.1095/biolreprod51.4.739

Figure Lengend Snippet: FIG. 1. Immunofluorescence of CD44 in human endometrium. a) Proliferative phase tissue (mAb P3H9) showing immunoreactivity in epithelial cells in a gland as well as in surrounding stroma. b) Decidua of first trimester (mAb P3H9) with strong reactivity in decidual stromal cells. c) Late secretory phase tissue (mAb PIG12) showing lateral staining in gland cells. Gland lumen is to right of the field. d) Nuclear staining in same sections as c. e) Example of tissue (late secretory phase) in which stromal reactivity is present but many glands are unstained (mAb PIG12). Note that one gland at bottom right is immunopositive. f) Nuclear staining, same field as (e). Arrowheads in a, c, and e indicate the apical cell surface of glandular epithelium.

Article Snippet: MATERIALS AND METHODS Antibodies Mouse pan anti-CD44 monoclonal antibodies (mAbs) were as follows: P1G12 and P3H9 (14,15; Chemicon, Temecula, CA); F10-44-2 (Serotec, Oxford, UK); BU52 (The Binding Site, Birmingham, UK); A1G3 (Seralab, High Wycombe, UK); and E1.2, a generous gift from Dr. C. Isacke, Imperial College, London [38].

Techniques: Immunofluorescence, Staining

Journal: Biology of reproduction

Article Title: Expression of two isoforms of CD44 in human endometrium.

doi: 10.1095/biolreprod51.4.739

Figure Lengend Snippet: FIG. 2. Immunofluorescence of CD44 in endometrial epithelial cells (mAb P3H9). al) Freshly isolated gland fragment in confocal microscopy. b) Pri- mary culture at 4 days showing mainly lateral immunoreactivity. c) Ishi- kawa endometrial adenocarcinoma cells with lateral as well as punctate surface staining.

Article Snippet: MATERIALS AND METHODS Antibodies Mouse pan anti-CD44 monoclonal antibodies (mAbs) were as follows: P1G12 and P3H9 (14,15; Chemicon, Temecula, CA); F10-44-2 (Serotec, Oxford, UK); BU52 (The Binding Site, Birmingham, UK); A1G3 (Seralab, High Wycombe, UK); and E1.2, a generous gift from Dr. C. Isacke, Imperial College, London [38].

Techniques: Immunofluorescence, Isolation, Confocal Microscopy, Staining

Journal: Biology of reproduction

Article Title: Expression of two isoforms of CD44 in human endometrium.

doi: 10.1095/biolreprod51.4.739

Figure Lengend Snippet: FIG. 3. Immunoprecipitation of CD44 from surface iodinated gland cells with mAb PIG12 gives a band at approximately 130 kDa. Right lane: con- trol, irrelevant mAb. Dots at right indicate positions of molecular size mark- ers (from the top down) of 200, 116, 67, and 45 kDa.

Article Snippet: MATERIALS AND METHODS Antibodies Mouse pan anti-CD44 monoclonal antibodies (mAbs) were as follows: P1G12 and P3H9 (14,15; Chemicon, Temecula, CA); F10-44-2 (Serotec, Oxford, UK); BU52 (The Binding Site, Birmingham, UK); A1G3 (Seralab, High Wycombe, UK); and E1.2, a generous gift from Dr. C. Isacke, Imperial College, London [38].

Techniques: Immunoprecipitation