Fig 1: The effects of pentraxin 3 (PTX3) antibodies on triple-negative breast cancer (TNBC) cells. (A) PTX3 antibodies (Ab-10 or Ab-49) inhibit the interaction between PTX3 and CD44. The abilities of PTX3- and PTX3 pre-incubated with Ab-10 or Ab-49 to bind immobilised CD44 were assessed using a competitive binding assay. (B) Epitope identification by ELISA using the indicated PTX3 antibodies and immobilised PTX3 or various shortened PTX3 peptides; the PTX3 amino acids 200–236 (RI37), 320–359 (GI40), 209–217 (AD9) and 352–360 (GR9) were examined for recognition by Ab-10 or Ab-49. (C) The suppressive effect of PTX3 antibodies on PTX3-activated signalling pathways in MDA-MB-231 cells. MDA-MB-231 cell lysates were incubated with isotype antibody (IgG1?), Ab-10 or Ab-49 and harvested for immunoblotting with the indicated antibodies. (D) Quantitative analysis of the protein activity of p-AKT, p-p65 and p-ERK1/2 in MDA-MB-231 cells. (E) The suppressive effect of PTX3 antibodies on PTX3-activated gene transcription in MDA-MB-231 cells. The total RNA of MDA-MB-231 cells that were incubated with IgG1?, Ab-10 or Ab-49 was harvested for real-time reverse transcription polymerase chain reaction (RT-PCR). (F) The migration and invasion of IgG1?-, Ab-10- and Ab-49-treated MDA-MB-231 cells with or without PTX3 treatment were assessed by Transwell assay. (G) An in vitro sphere formation assay was performed with IgG1?-, Ab-10- and Ab-49-treated MDA-MB-231 cells with or without PTX3 treatment. (H) IgG1?, PTX3 antibody (Ab-10) and paclitaxel (Taxol) in the indicated groups of orthotopically allografted 4T1-Luc2-bearing mice by intraperitoneal injection. (I) The effects of IgG1?, Ab-10 and Taxol on the growth of 4T1-Luc2 tumours in BALB/c mice were measured using external calipers. (J) Representative in vivo bioluminescence images, (K) metastasis quantification of 4T1-Luc2 tumours in lung and brain and (L) Kaplan–Meier survival curves after administering IgG1?, PTX3 antibody (Ab-10) or paclitaxel (Taxol) in the indicated groups of 4T1-Luc2-bearing mice. All data are expressed as the mean ± SEM. Differences among groups were analysed using one-way ANOVA followed by Tukey's multiple comparison test. *p < .05, **p < .01, ***p < .001, ns: no significance
Fig 2: A comparable decrease in A549 cell viability is observed upon using either glycosylated or nonglycosylated IGFBP-3 protein. IGFBP-3 protein glycosylated (Gly) or nonglycosylated (Non-Gly) was added (50 nm) to cells in the absence or presence of the CD44 antibody, 5F12, known to block HA-CD44 interactions. Cell viability was assessed by the MTT assay. Cells were seeded in 96-well plates at 0.2 × 105 cells per well in 10% FBS-supplemented media. The following day, the cell monolayers were incubated in serum-free medium for 12 h and then treated as indicated for 48 h with the media containing the specific components in the different treatments replaced every 12 h. The CD44 antibody (5 µg·mL-1) was added either separately or 2 h prior to addition of IGFBP-3 proteins. Optical density measurements (570 nm) were normalized by expressing each point in relation to the untreated control of each cell line (set to 100%). Each column represents the mean ± SD of three independent experiments, each performed in triplicate. Asterisks (*) indicate a statistically significant difference from the corresponding untreated cell line control, *P < 0.05, **P < 0.01 of each cell line. The absence of asterisks indicates no significance, Mann–Whitney test.
Fig 3: Schematic representation of the main findings of this study. Binding of IGFBP-3 to either humanin (HN) or to the glycosaminoglycan, hyaluronan (HA), is not affected by either glycosylation or reduction. Glycosylated and reduced CD44 is less able than de-N-glycosylated and oxidized CD44 to compete with IGFBP-3 for binding HA.
Fig 4: CHI3L1 binds to CD44 which also interacts with IL-13Ra2. a Western blot analysis of CHI3L1, CD44 and IL-13Ra2 protein expression in various gastric cancer cell lines. b-c Lysates from AGS and MGC803 cells were immunoprecipitated (IP) with control IgG and anti-CD44 or anti-CHI3L1 antibody, and then immunoblotted as indicated. Five percent of total cell lysates were used for the input. d Co-localization of CD44 (green) and CHI3L1 (red) in AGS (upper panel), MGC803 (middle panel) and GC tissues from patient #1 (lower panel) by immunofluorescent confocal microscopy (Magnification: 630×). Scale bars represent 10 µm. e-f Lysates from AGS and MGC803 cells were immunoprecipitated (IP) with IgG and anti-CD44 or anti-IL-13Ra2 antibody, and then immunoblotted as indicated. Five percent of total cell lysates were used for the input. g Co-localization of CD44 (green) and IL-13Ra2 (red) in AGS (upper panel), MGC803 (middle panel) and GC tissues from patient #2 (lower panel) by immunofluorescent confocal microscopy (Magnification: 630×). Scale bars represent 10 µm
Fig 5: CHI3L1 triggers Erk and Akt signaling through CD44. a and b Western blot analysis of Erk and Akt activation in the AGS and MGC803 cells which were pre-exposed with the control antibody or functional CD44 blocking antibody and treated with rhCHI3L1 (500 ng/ml) for the indicated times. c and d Western blot analysis of CD44 and IL-13Ra2 knockdown efficacy in AGS (c) and MGC803 (d) transfected with scramble, CD44 or IL-13Ra2 shRNA. e and f Westen blot evaluation of the activation of Erk and Akt in AGS and MGC803 cells which stably expressing scrambled, CD44 or IL-13Ra2 shRNA and were cultured in the presence or absence of rhCHI3L1 (500 ng/ml). g Westen blot analysis of the activation of Erk and Akt in bone marrow-derived macrophages (BMDM) from WT and CD44-/- mice which were incubated with the recombinant mouse CHI3L1 (500 ng/ml) for the noted periods of time
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