Fig 2: Detection of FPR1 expression on Tissue Micro Array, frozen sections and qPCR. Bar histogram representing results obtained from a Tissue MicroArray (TMA). Immunohistochemical detection of FPR1 (ab#150533) expression on formalin fixed-paraffin embedded GBM patient specimens. Semi-quantitative evaluation was performed by averaging the scores of 4 cores derived from each patient specimen and plotted with a corresponding bar. The intensity of FPR1 expression was scored on a scale from 0 to 3; 0 being negative, 1 positive but with focal and diffuse staining, 2 prevalently focal and more intense staining and 3 exhibiting highly intense focal staining. On average patient samples exhibited an intensity score of 2. All samples were positive for FPR1 (a). Representative pictures of 4 different core biopsies containing FPR1 intensities from 0 to 3 (b). Immunohistochemical detection of FPR1 (ab#101659) expression on 36 frozen GBM patient samples and quantification containing on average 32 ± 14 % FPR1 positive cells (c). Representative FPR1 immunohistochemical staining on a frozen GBM section (d). FPR1 mRNA detection on GBM snap frozen tissue samples by qPCR. All samples were loaded in 4 replicates, FPR1 mRNA values varied from minimum (2−ΔCT = 9.34 × 10−4) to maximum (2−ΔCT = 1.1 × 10−1) (e)

Fig 3: Immuno -hisotochemical and -fluorescence detection of FPR1 (ab#150533) alone or coupled with GFAP and CD68/CD163 markers. Representative photomicrographs depicting immunohistochemical expression of FPR1 in the GBM patient specimens and its derived primary GBM cell line. The original patient specimens all displayed FPR1 expression with variable intensities. Images of patient GBM tissue of which GG cell lines were obtained (a–d). Immunofluorescence image displaying GFAP single expression (arrow) and co-expression with FPR1 (arrow head) (panel e). FPR1 shows single expression (asterisk) and co-expression with CD68 (arrow head) (panel f). Single stained FPR1 cells (asterisk) and co-expression with CD163 (arrow head) (panel g)

Fig 4: Inhibition of mitochondrial induced FPR1 activity by CHIPS in U87 cells. Mitochondrial peptides fMLKLIV and fMMYALF induce dose dependent calcium mobilization (10−6 M–10−8 M) of U87 cells (a, c), which can be dose dependently inhibited with 0.01–10 μg/mL CHIPS (b, d). In U87 cells 10−6 M fMLKLIV or fMMYALF induced AKT phosphorylation on the Ser473 site at 5, 15 and 30 min. At the same time points 10 μg/mL CHIPS showed 46 ± 33, 52 ± 7, 67 ± 12 % inhibition of fMLKLIV induced phosphorylation and respectively 67 ± 39, 78 ± 29, 70 ± 40 % inhibition of fMMYALF-induced phosporylation (e, f). A concentration of 10−6 M fMLKLIV or fMMYALF induce ERK1/2 phosphorylation in U87 cells at 5, 15 and 30 min. When U87 cells were pre-treated with CHIPS (10 μg/mL), fMLKLIV induced phosphorylation was inhibited up to 100 % and fMMYALF-induced phosphorylation was inhibited 94 ± 16, 99 ± 1 and 92 ± 12 % (respectively at time points 5, 15 and 30 min) (g and h). For migration in transwell assays each time the cell migration towards one of the ligands was set at 100 % and inhibition with CHIPS was plotted against it. Migration towards fMLF, fMMYALF and fMLKLIV of U87 cells was inhibited up to 42 ± 14 % (P = 0.018), 34 ± 27 % (P = 0.028) and 36 ± 29 % (P = 0.028) when pretreated with 10 μg/mL CHIPS. Values are indicated as mean ± SD (i)