PMC

A. VeraTag FFPE quantification of HGF in unstimulated and HGF stimulated A549 cells. Isotype IgG control signals (inset) were subtracted from the HGF signals. Bi. VeraTag quantification of HGF in Ln18, Ln229, U138, U118, and U87MG glioma cell lines. Isotype IgG control signals (inset) were subtracted from HGF assay signals. ND: Not detectable. Bii. ELISA determinations of HGF in A549, Ln18, Ln229, U138, U118, and U87MG cell lysates. ND: Not detectable. Ci. VeraTag FFPE quantification of HGF in HEK293/HGF (clone 1) and U138 glioma cell lines. Cii. IHC detection of HGF in HEK293/HGF stable clone (clone 1) and U138 glioma cells.

The effect of YangZheng XiaoJi and cMET kinase inhibitors, PHA665752 and SU11274 on HGF-induced cell migration (A, B, C, D) and invasion (a, b, c, d), on both SKMES1 (top panel) and A549 (bottom panel) cells. Migration assay A both PHA665752 (10 nM) and SU11274 (20 nM) had a significant effect on HGF-induced migration of SKMES1 cells. HGF concentration shown here is 50 ng/ml. *p < 0.01 cells with HGF/PHA665752 combination vs cells with control medium only; **p < 0.05 cells with HGF/SU11274 combination vs cells with HGF only. ^Ɵp = 0.059 cells with HGF alone vs cells with control medium. B The effect of the combination between DME25 and HGF inhibitors on SKMES1 cells. *p < 0.05 cells with HGF/SU11274 (or HGF/PHA665752) combination vs cells with HGF only; **p < 0.05 cells with HGF/SU11274/DME25 (or HGF/PHA665752/DME25) combination vs cells with HGF only; ^#p < 0.05 cells with HGF/PHA665752 combination vs cells with control medium only; ^Ɵp = 0.059 cells with HGF alone vs cells with control medium. C effect of HGF and HGF receptor inhibitors on the migration of A549 cells. *p < 0.01 cells with HGF/SU11274 (or HGF/PHA665752) combination vs cells with HGF only; ^#p < 0.05 respective cells vs cells with control medium only. D effect of HGF, and the combination of DME25 and HGF receptor inhibitors on the migration of A549 cells. *p < 0.01 cells with HGF/SU11274 (or HGF/PHA665752) combination vs cells with HGF only; **p < 0.05 cells with HGF/SU11274/DME25 (or HGF/PHA665752/DME25, or HGF/DME25) combination vs cells with HGF only; ^#p < 0.05 respective cells vs cells with control medium only. Invasion assay a, b DME25 significantly potentiated the effect of HGF receptor inhibitors, PHA665752 (a) and SU121274 (b), on HGF-induced invasion of SKMES1 cells. The effects were almost identical on both compounds. *p < 0.01 treatment plus DME25 vs respective treatment without DME25 control; **p < 0.05 treatment plus DME25 vs respective treatment without DME25 control; ^#p < 0.05 respective cells vs cells with control medium only. c, d Effects of DME25 on HGF and HGF receptor inhibitors, PHA665752 (c) and SU121274 (d), on HGF-induced invasion of A549 cells. *p < 0.01 treatment plus DME25 vs respective treatment without DME25 control; **p < 0.05 treatment plus DME25 vs respective treatment without DME25 control; # p < 0.05 respective cells vs cells with control medium only. The concentrations use for the test agents were: PHA665752—10 nM, SU11274—20 nM, HGF—50 ng/ml and DME25—1:1000 dilution

Serum and peritoneal fluid concentrations of HGF and its gene and protein expression by PFMCs, PBMCs and ESCs. Serum concentrations of HGF were measured in 70 endometriotic and 70 non-endometriotic participants. Peritoneal fluid concentrations of HGF were measured in 36 endometriotic and 30 non-endometriotic participants. The basal gene and protein expression of HGF were measured in PFMCs (n = 10), PBMCs (n = 10), EESCs (n = 8), and EuESCs (n = 10) from patients with endometriosis and PFMCs (n = 7), PBMCs (n = 10), and CESCs (n = 10) from non-endometriotic women. (Aa) serum concentration of HGF, (Ab) peritoneal concentration of HGF, (Ac) serum concentration of HGF in different stages of endometriosis, (Ad) serum concentration of HGF in different menstrual cycles, (Ba) HGF gene expression by PFMCs, (Bb) HGF protein expression by PFMCs, (Bc) HGF gene expression by PBMCs, (Bd) HGF protein expression by PBMCs, (Be) HGF gene expression by ESCs, (Bf) HGF protein expression by ESCs. *P < 0.05, **P < 0.01, ***P < 0.001. A and B parts analyzed by parametric and non-parametric tests, respectively. ^†P–C: Proliferative phase of the control group, S-C: Secretory phase of the control group, P-E: Proliferative phase of endometriosis patients, S–E: Secretory phase of endometriosis patients

(A, B) Fibroblasts isolated from young and aged human skin were similarly expressed HGF mRNA (A) and protein (B). Human skin dermal fibroblasts were prepared from young (mean age 27±1 years, N=6) and aged (mean age 83±1.4 years, N=6) buttock skin (see for details). (A) HGF mRNA levels were quantified by real-time RT-PCR. Mean±SEM, N=6. (B) HGF protein levels were quantified by ProteinSimple capillary electrophoresis immunoassay (see for details). Insert shows representative digital images. Mean±SEM, N=6. (C) Elevated expression of HGF in aged human skin in vivo. Fibroblasts were captured from young (22–30 years) and aged (80+ years) individuals by laser capture microdissection (see for details). HGF mRNA levels were quantified by real-time RT-PCR. Mean±SEM, N=6, *p<0.05. (D, E) Presenescent and senescent dermal fibroblasts were similarly expressed HGF mRNA (D) and protein (E). (D). Total RNA was prepared from pre-senescent and senescent dermal fibroblasts and HGF mRNA levels were quantified by real-time RT-PCR. Mean±SEM, N=6. (E) Whole cell protein extract was prepared from pre-senescent and senescent dermal fibroblasts and HGF protein levels were quantified by ProteinSimple capillary electrophoresis immunoassay (see for details). Insert shows representative digital images. Mean±SEM, N=6. HGF mRNA levels (A, C, D) were normalized to the housekeeping gene 36B4, as an internal control for quantification. HGF protein levels (B, E) were normalized by β-actin (loading control).