Fig 1: ESM1 overexpression promoted EC cell invasion and angiogenesis, while silencing of ESM1 had opposite effects. (a–d) The invasion of AN3CA or RL95-2 cells in the control, shNC, short hairpin RNA-targeted ESM1 (shESM1-1), NC, and ESM1 groups was detected by Transwell assay. The representative image was presented (magnification: ×250 times; scale bar: 50 µm) and relative invasion rate was quantified. (e–h) Tube formation assay was performed to assess the effect of ESM1 overexpression or silencing on angiogenesis. The representative image was presented (magnification: ×100 times; scale bar: 50 µm) and angiogenesis length was measured. *** P < 0.001 vs Sh-NC; # P < 0.05, ### P < 0.001 vs NC.
Fig 2: Endocan levels according to renal allograft status. (A) Plasma endocan levels were significantly higher in patients with acute ABMR than those in patients with NP, ATN, APN, BKVN, and TCMR. Patients with chronic active ABMR exhibited significantly higher plasma endocan levels than patients with LTGS. (B) Similar to plasma endocan levels, urinary endocan levels were significantly increased in patients with acute ABMR compared to those in the other groups in the short transplant vintage set. Patients with TCMR exhibited higher urinary endocan levels than patients with NP, and the levels were also higher in patients with chronic active ABMR than in patients with LTGS. *p < 0.05, **p < 0.005. Abbreviations: NP, normal pathology; ATN, acute tubular necrosis; APN, acute pyelonephritis; BKVN, BK virus associated nephropathy; TCMR, T-cell mediated rejection; aABMR, acute antibody-mediated rejection; LTGS, long-term graft survival; cABMR, chronic active antibody-mediated rejection.
Fig 3: SPI1 overexpression promoted ESM1 level and viability of EC cells, and silencing of ESM1 reversed the effect of SPI1 overexpression on the viability of EC cells. (a) The expression of SPI1 in EMCs as well as AN3CA and RL95-2 cells was detected by qRT-PCR, and SPI1 was highly expressed in AN3CA and RL95-2 cells. (b and c) After AN3CA and RL95-2 cells were transfected with SPI1 overexpression plasmid, the expression levels of SPI1 and ESM1 were quantified by qRT-PCR. (d and e) Twenty-four, forty-eight, or seventy-two hours after cell transfection with SPI1 overexpression plasmid and shESM1, the effects of overexpressed SPI1 and silent ESM1 on the viability of AN3CA and RL95-2 cells were evaluated by MTT assay. ^^^ P < 0.001 vs EMC; &&& P < 0.001 vs NC; * P < 0.05, ** P < 0.01, *** P < 0.001 vs NC + ShNC; # P < 0.05, ## P < 0.01, ### P < 0.001 vs SPI1 + ShNC.
Fig 4: ESM1 was induced by SPI1, which was highly expressed in EC. (a) The motif logo of SPI1 was analyzed by JASPAR (http://jaspar.genereg.net/analysis). (b) The ESM1-binding sites with the SPI1 promoters. (c) SPI1 bound to ESM1, which was verified by dual-luciferase reporter assay. (d) The expression of SPI1 in the cancer tissues and adjacent tissues of 64 EC patients was quantified by qRT-PCR, and SPI1 was highly expressed in EC tissues. (e) The correlation between SPI1 expression and ESM1 in EC tissues was analyzed by Pearson test, the results of which indicated that SPI1 was positively related to ESM1. *** P < 0.001 vs EC tissue; ### P < 0.001 vs NC.
Fig 5: Expression of ESM1 in EC cells. (a) qRT-PCR was applied to quantify the expression of ESM1 in EMCs and EC cells (HEC-1B, HEC-1A, AN3CA, and RL95-2). (b and c) After AN3CA cells were transfected with shESM1-1, shESM1-2, or ESM1 overexpression plasmid, the level of ESM1 in AN3CA cells was determined by qRT-PCR. (d and e) After RL95-2 cells were transfected with shESM1-1, shESM1-2, or ESM1 overexpression plasmid, the level of ESM1 in RL95-2 cells was determined by qRT-PCR. GAPDH was applied as a standardized gene. *** P < 0.001 vs EMC; ### P < 0.001 vs short hairpin RNA-targeted negative control (shNC); ^^ P < 0.01, ^^^ P < 0.001 vs NC.
Supplier Page from Abcam for Anti-ESM1 antibody