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

Journal: International journal of oncology

Article Title: Epithelial membrane protein 3 regulates lung cancer stem cells via the TGF‑β signaling pathway.

doi: 10.3892/ijo.2021.5261

Figure Lengend Snippet: Figure 2. EMP3 regulates CSC properties in lung CSC. (A) Western blot analysis of the CSC marker proteins CD133, ALDH1A1, ALDH1A3 and CD44. The CSC regulatory proteins Sox2, Oct‑4 and Nanog were also analyzed by western blotting. A549 cells were transfected with siRNA targeting EMP3. (B) Immunocytochemistry analysis of CSC marker proteins using siRNA treated A549 cells. The green signal is produced by the GFP tag on the secondary antibody. (C) Sphere‑forming capacity analysis of A549 cells in siRNA transfected EMP3 cells and (D) the ability to of an anti‑EMP3 antibody to abrogate sphere formation was assessed. (E) Single‑cell assay of si‑EMP3 transfected cells and (F) the ability of the anti‑EMP3 antibody to abrogate this. (G) Limiting dilution assays were performed in 96 well plates. Wells were plated with 1, 50, 100, 150 or 200 cells/well, and conditioned media was added. Results were confirmed 10 days after seeding of cells. Colony‑formation assays to observe the clonogenicity of the (H) EMP3‑knockdown A549 and (I) anti‑EMP3 antibody treated A549 cells. Cells were irradiated with 3 Gy radiation. After 10 days of incubation, the colonies were stained with crystal violet. (J) Western blot analysis of members of the Sonic hedgehog, Wnt/β‑catenin and Notch signaling pathways in CSCs. Data are presented as the mean ± standard deviation of three repeats. Scale bar, 50 µm. *P<0.05 vs. control. EMP3, epithelial membrane protein 3; ALDH1, aldehyde dehydrogenase 1; CSC, cancer stem cell; Sox2, sex determining region Y‑box 2; Oct‑4, octamer‑binding transcription factor 4; siRNA, small interfering RNA; p, phosphorylated; GSK3‑β, glycogen synthase kinase 3‑β.

Article Snippet: Antibodies against EMP3 (cat. no. ab236671; Abcam), Sox2 (sex determining region Y‐box 2; cat. no. 3579; Cell Signaling Technology, Inc.), phos‐ phorylated (p)‐Smad2 (cat. no. 18338; Cell Signaling Technology, Inc.), p‐Smad3 (cat. no. 9520; Cel l Signaling Technology, Inc.), Smad2/3 (cat. no. 5678; Cell Signaling Technology, Inc.), β‐actin (cat. no. sc‐7963; Santa Cruz Biotechnology, Inc.), TGFBR1 (cat. no. sc‐518086; Santa Cruz Biotechnology, Inc.), TGFBR2 (cat. no. sc‐17791; Santa Cruz Biotechnology, Inc.), TGFBR3 (cat. no. sc‐74511; Santa Cruz Biotechnology, Inc.), CD44 (cat. no. 5640; Cell Signaling Technology, Inc.), β‐catenin (cat. no. sc‐7963; Santa Cruz Biotechnology, Inc.), Twist (cat. no. sc‐15393; Santa Cruz Biotechnology, Inc.), Vimentin (MA5‐16409; Thermo Fisher Scientific, Inc.), alde‐ hyde dehydrogenase (ALDH1)A1 (cat. no. ab52492; Abcam), ALDH1A3 (cat. no. ab129815; Abcam), Snail (cat. no. sc‐10432; Santa Cruz Biotechnology, Inc.), Slug (cat. no. sc‐166476; Santa Cruz Biotechnology, Inc.), ZEB1 (Zinc finger E‐box‐binding homeobox 1; cat. no. sc‐25388; Santa Cruz Biotechnology, Inc.), E‐cadherin (cat. no ab15148; Abcam), N‐cadherin (cat. no. 610921; BD Biosciences) and Oct4 (cat. no. 2750; Cell Signaling Technology, Inc.) were used.

Techniques: Western Blot, Marker, Transfection, Immunocytochemistry, Produced, Irradiation, Incubation, Staining, Protein-Protein interactions, Standard Deviation, Control, Membrane, Small Interfering RNA

Journal: International journal of molecular sciences

Article Title: Inhibiting CD44-ICD Attenuates LPS-Induced Initiation of Hepatic Inflammation in Septic Mice.

doi: 10.3390/ijms25168907

Figure Lengend Snippet: Figure 1. Effects of inhibiting CD44-ICD on LPS-induced hepatic inflammation and histological change in mice. Twenty mice were divided into four groups. N group mice were given only saline; D group mice were given DAPT alone; L group mice were given LPS alone; LD group mice were given DAPT 30 min before LPS. After 6 h, (A) AST levels, (B) ALT levels in serum, and (C) liver histopathological changes (10×, scale bar—100 µm; magnified 20×, scale bar—50 µm) by H&E staining were determined. Data are expressed as mean ± SD (n = 5). * p < 0.05 compared with N and D groups. # p < 0.05 compared with the L group.

Article Snippet: After blocking, the blots were incubated with iNOS (1:1000; BD Bioscience, Franklin Lakes, NJ, USA, 610432), p-IKK α/β (1:1000; Genetex, Washington, DC, USA, GTX9039), p-IκB (1:1000; Genetex, GTX00967), nuclear NF-κB (1:500; Genetex, GTX102090), IL-1β (1:1000, Santacruz, Dallas, TX, USA, sc-127420), CD44 (1:500; proteintech, 15675-1-AP), nuclear CD44-ICD (1:500; Cosmobio, Tokyo, Japan, #KAL-K0604), GAPDH (1:5000; abcam, ab9485), β tubulin (1:1000; abcam, ab6046), and Lamin B1 (1:1000; abcam, ab16048) in 5% nonfat skim milk.

Techniques: Saline, Staining

Journal: International journal of molecular sciences

Article Title: Inhibiting CD44-ICD Attenuates LPS-Induced Initiation of Hepatic Inflammation in Septic Mice.

doi: 10.3390/ijms25168907

Figure Lengend Snippet: Figure 2. Effects of inhibiting CD44-ICD on pro-inflammatory mediators in LPS-induced hepatic inflammation in mice and cells. Twenty mice were divided into 4 groups. N group mice were given only saline; D group mice were given DAPT alone; L group mice were given LPS alone; LD group mice were given DAPT 30 min before LPS. (A) hepatic IL-1β, (B) NO, and (C) iNOS expressions were determined six hours after LPS. The Chang cells were divided into five groups. N group, cells were treated only DMSO; L group, cells received LPS (100 ng/mL); LD5 group, cells received 5 mM DAPT 1 h before LPS; LD10 group, received 10 mM DAPT 1 h before LPS; LD 20 group, cells were received 20 mM DAPT 1 h before LPS. (D) IL-1β levels in Chang cells were determined 6 h after LPS was given. Data are expressed as mean ± SD (n = 5). * p < 0.05 compared with N and D groups. # p < 0.05 compared with the L group.

Article Snippet: After blocking, the blots were incubated with iNOS (1:1000; BD Bioscience, Franklin Lakes, NJ, USA, 610432), p-IKK α/β (1:1000; Genetex, Washington, DC, USA, GTX9039), p-IκB (1:1000; Genetex, GTX00967), nuclear NF-κB (1:500; Genetex, GTX102090), IL-1β (1:1000, Santacruz, Dallas, TX, USA, sc-127420), CD44 (1:500; proteintech, 15675-1-AP), nuclear CD44-ICD (1:500; Cosmobio, Tokyo, Japan, #KAL-K0604), GAPDH (1:5000; abcam, ab9485), β tubulin (1:1000; abcam, ab6046), and Lamin B1 (1:1000; abcam, ab16048) in 5% nonfat skim milk.

Techniques: Saline

Journal: International journal of molecular sciences

Article Title: Inhibiting CD44-ICD Attenuates LPS-Induced Initiation of Hepatic Inflammation in Septic Mice.

doi: 10.3390/ijms25168907

Figure Lengend Snippet: Figure 3. Effects of inhibiting CD44-ICD on NF-κB signaling-related protein expressions in LPS- induced hepatic inflammation in mice. Twenty mice were divided into 4 groups. N group mice were given only saline; D group mice were given DAPT alone; L group mice were given LPS alone; LD group mice were given DAPT 30 min before LPS. (A) Western blotting analysis of p- IKK, (B) immunohistochemical staining analysis of p-IKK, (C) Western blotting analysis of p-IκB, (D) immunohistochemical staining analysis of p-IκB, and (E) Western blotting analysis of nuclear NF-κB expression in liver tissue was determined 6 h after LPS. The immunohistochemical positive expression were observed under a microscope (10× with a scale bar of 100 µm). Data are expressed as mean ± SD (n = 5). * p < 0.05 compared with N and D groups. # p < 0.05 compared with the L group.

Article Snippet: After blocking, the blots were incubated with iNOS (1:1000; BD Bioscience, Franklin Lakes, NJ, USA, 610432), p-IKK α/β (1:1000; Genetex, Washington, DC, USA, GTX9039), p-IκB (1:1000; Genetex, GTX00967), nuclear NF-κB (1:500; Genetex, GTX102090), IL-1β (1:1000, Santacruz, Dallas, TX, USA, sc-127420), CD44 (1:500; proteintech, 15675-1-AP), nuclear CD44-ICD (1:500; Cosmobio, Tokyo, Japan, #KAL-K0604), GAPDH (1:5000; abcam, ab9485), β tubulin (1:1000; abcam, ab6046), and Lamin B1 (1:1000; abcam, ab16048) in 5% nonfat skim milk.

Techniques: Saline, Western Blot, Immunohistochemical staining, Staining, Expressing, Microscopy

Journal: International journal of molecular sciences

Article Title: Inhibiting CD44-ICD Attenuates LPS-Induced Initiation of Hepatic Inflammation in Septic Mice.

doi: 10.3390/ijms25168907

Figure Lengend Snippet: Figure 4. Effects of inhibiting CD44-ICD on CD44 expression in LPS-induced hepatic inflammation in mice and cells. Twenty mice were divided into 4 groups. N group mice were given only saline; D group mice were given DAPT alone; L group mice were given LPS alone; LD group mice were given DAPT 30 min before LPS. (A) Western blotting analysis of hepatic CD44 expression and (B) immunofluorescence staining analysis of hepatic CD44 expression was determined 6 h after LPS. Positive immunofluorescence reaction for CD44 (red color) and DAPI nucleic acid staining (blue color) was observed (20×) under a microscope. The Chang cells were divided into five groups. N group, cells were treated with only DMSO; L group, cells received LPS (100 ng/mL); LD5 group, cells received 5 mM DAPT 1 h before LPS; LD10 group received 10 mM DAPT 1 h before LPS; LD 20 group, cells received 20 mM DAPT 1 h before LPS. (C) CD44 and (D) nuclear CD44-ICD expression in Chang cells were determined 6 h after LPS was given. Data are expressed as mean ± SD (n = 5). * p < 0.05 compared with N and D groups. # p < 0.05 compared with the L group.

Article Snippet: After blocking, the blots were incubated with iNOS (1:1000; BD Bioscience, Franklin Lakes, NJ, USA, 610432), p-IKK α/β (1:1000; Genetex, Washington, DC, USA, GTX9039), p-IκB (1:1000; Genetex, GTX00967), nuclear NF-κB (1:500; Genetex, GTX102090), IL-1β (1:1000, Santacruz, Dallas, TX, USA, sc-127420), CD44 (1:500; proteintech, 15675-1-AP), nuclear CD44-ICD (1:500; Cosmobio, Tokyo, Japan, #KAL-K0604), GAPDH (1:5000; abcam, ab9485), β tubulin (1:1000; abcam, ab6046), and Lamin B1 (1:1000; abcam, ab16048) in 5% nonfat skim milk.

Techniques: Expressing, Saline, Western Blot, Immunofluorescence, Staining, Microscopy