Fig 2: Establishment of the AEPC purification. (A) Schematic view of the AEPC purification method from human lipoaspirates. AEPCs were purified from the SVF using two MACS sorting steps (CD45-CD31+ sorting and only CD31+ sorting) and adherent cultures. (B,C) Optimization of the timing for the second MACS sorting step for AEPC purification. Cells were dissociated from the cell culture surface with TrypLE Express Enzyme after either 4.5 or 7 days of culture and were subjected to MACS separation using CD31 microbeads. Cell surface markers (CD45, CD31, and CD34) were examined by flow cytometry. The percentage of CD45-CD31+ (AEPC) cells in CD45-CD31- (ASCs, isotype+ cells, other cells, and debris) cells is shown in magenta. ASCs drastically decreased CD34 expression in vitro, which could be observed in the CD45-CD31- population (arrowheads). Scale bars in microscopic photographs represent 100 µm. This experiment was performed twice, independently from 2 donors. (D) Morphologies of purified AEPCs over time. The photographs were taken using a phase-contrast microscope (Leica DM IL LED with a camera MC170HD; 100× magnification). P1D6 indicates the culture period at passage 1, after 6 days in culture. The experiment was performed twice, independently from 2 donors. Bars represent 100 µm.

Fig 3: Effects of breast milk on lipid accumulation in 3T3-L1 adipocytes. Preadipocytes were differentiated for eight days in the presence or absence of breast milk (BM) whey, and oil red O staining was performed on day 8. Stained adipocytes were viewed using a Leica DMIL LED Tissue Culture Microscope, and images were taken using the Infinity Camera Analyze Software at 400× magnification. Scale bar = 50 µm (A) Relative lipid droplet size analyzed using the ImageJ software. Values are expressed as mean ± SD, n = 3, different superscripts (a, b, c) are used to denote significant differences between the treatment groups, p < 0.05 was considered significant (B) Representative images of the cells stained with oil red O. Q—quartile.

Fig 4: Forssman antigen expression of COS1(B3GALNT1) cells transfected with the original and amino acid substitution constructs of mouse GBGT1 gene, mouse ABO-AB gene, and human ABO-A gene.Results of anti-Forssman antibody immunostaining of COS1(B3GALNT1) cells after DNA transfection are shown. Cells were imaged in color using a Leica DMIL LED microscope equipped with D-LUX3 camera. All images were taken at the same magnification using a 10x/0.25 PH1 objective. Figures were created using Adobe Photoshop software. The eukaryotic expression constructs prepared in the pSG5 plasmid vector were used. The results are: mouse original GBGT1 gene (M_GBGT1) having GlyGlyAla tripeptide and its in vitro mutagenized amino acid substitution constructs with either LeuGlyGly or MetGlyAla tripeptide in the top row, mouse original ABO-AB gene (M_ABO-AB) with GlyGlyAla tripeptide and its substitution constructs having LeuGlyGly or MetGlyAla in the middle row, and human A allele of ABO gene (H_ABO-A) with LeuGlyGly tripeptide and its substitution constructs having GlyGlyAla or MetGlyAla tripeptide in the bottom row. Under the experimental conditions used, cells became confluent and formed a monolayer. Strong positive staining with anti-Forssman antibody was observed by DNA transfection of original M_GBGT1 and M_ABO-AB expression constructs. Additionally, COS1(B3GALNT1) cells transfected with H_ABO-A(GGA), the H_ABO-A construct with GlyGlyAla tripeptide sequence, exhibited some positive staining. It should be noted that not all cells were stained positive by DNA transfection as opposed to the viral infection-FACS sorting results shown in Fig. 2. Bluish background color was due to DAB and nickel ions in color development solution.

Fig 5: Compound 145 significantly reduced TGF-ß1-induced MRC-5 transition into myofibroblasts. MRC-5 were pre-incubated for 1 h with the studied compounds (10 µM) and then cultured for 24 h (A, C) or 48 h (B, D–F) in TGF-ß1 (5 ng/mL). (A, C) qPCR was carried out to analyze ACTA2, MYH11, SM22, COL1A1, TNC, FN1, and VCAN transcript levels. Samples were run three times in duplicates. Cellular a-smooth muscle actin (a-SMA) (B) and collagen I (D) protein level was determined by in-cell ELISA; n = 8. (E) To visualize a-SMA positive stress fibers, MRC-5 were fixed, permeabilized, blocked, and labeled with anti-a-SMA antibody, followed by Alexa Fluor 488 conjugated antibody, nuclei were stained with Hoechst 33342 dye. Microphotographs were taken using a Leica DMiL LED Fluo microscope, 40× objective, bar = 50 µm. (F) Myofibroblasts (MRC-5 positive for a-SMA) were counted in 20 randomly selected fields of view and expressed as a percentage of the entire MRC-5 population. Each bar represents the mean value (± S.E.M.). The results were considered statistically significant at the p level of 0.05, against control (#) and TGF-ß1 (*).