Fig. 4.

Effects of Lactobacillus rhamnosus GG (LGG) supernatant (SN) on specialized proresolving mediator (SPM) biosynthetic machinery and angiogenic potential of colorectal carcinoma (CRC) cells. (A) ALOX5, ALOX15A, ALOX15B, BLT1, and ChemR23 protein expression levels (mean fluorescence intensity), assessed by cytofluorimetric analysis, in HT29 cells treated with Lactobacillus rhamnosus GG (LGG) supernatant (SN) or its control broth—1 : 30 titration for 3 h. A representative experiment of three independent experiments is shown. (B) Proresolving and proinflammatory autacoid (RvD1, LXB₄, LXA₄, PGE₂) release in HT29 and HCT116 cells treated with LGG SN—1 : 30 titration for 12 h over control (broth). Baseline values of each mediator were in HCT116 cells: RvD1 122 ± 15 pg/10⁶ cells, LXB₄ 38 ± 4.2 pg/10⁶ cells, LXA₄ 501 ± 54 pg/10⁶ cells and PGE₂ 101 ± 11 pg/10⁶ cells. Baseline values of each mediator were in HT29 cells: RvD1 128 ± 27 pg/10⁶ cells, LXB₄ 36 ± 5 pg/10⁶ cells, LXA₄ 475 ± 52 pg/10⁶ cells and PGE₂ 82 ± 9.8 pg/10⁶ cells. Data are represented as mean ± SD of changes over baseline levels obtained in five independent experiments. *P < 0.05 compared with the control by Student's t test. (C) VEGF‐A release in HCT116 and HT29 cells treated with LGG SN—1 : 30 titration or the control culture broth for 12 h. Data are represented as mean ± SD of five independent experiments. *P < 0.05 compared with the control by Student's t test. (D) Analysis of proteins in conditioned media (CM) from HT29 cells treated with LGG SN or its control broth (1 : 30 titration) using angiogenesis‐associated protein antibody arrays. The mean of protein pixel density for each angiogenesis‐related protein, normalized for the reference spots, was calculated and compared with the relative control. The array images and the relative quantitative profiles of protein levels are shown. (E) Human umbilical vein endothelial cells (HUVECs) were cultured in the presence of cell culture conditioned media (CM) from HCT116 or HT29 cells treated with LGG SN or the control culture broth (1 : 30 titration) (10× magnification) in a 24‐well plate. After 12 h, cells were fixed with ice‐cold 70% ethanol, and tubule formation was evaluated. Sample images and a quantification of the angiogenic response are reported. Scale bar 50 μm. Data are represented as mean ± SD of three independent experiments. *P < 0.05 compared with the control (broth) by Student's t test. [Colour figure can be viewed at wileyonlinelibrary.com]