Fig 1: Effects of SPRAC down-regulation on cell apoptosis. Cell apoptosis was detected using flow cytometry, Q1: cell debris, Q2: late apoptotic cells, Q3: normal growing cells, Q4: early apoptotic cells. (A) The total apoptosis rates of SPARC shRNA transfected and control shRNA-transfected cells in Ishikawa group were 16.3% (Q2:12.7%, Q4:3.6%) and 27.6% (Q2:20.0%, Q4:7.6%), separately. (B) The total apoptosis rates of SPARC shRNA transfected and control shRNA-transfected cells in HEC-1B group were 19.4% (Q2:14.1%, Q4:5.3%) and 27.4% (Q2:20.7%, Q4:6.7%), separately. (C) The apoptosis rate of SPARC shRNA-transfected cells was significantly inhibited after reducing the expression of SPARC in Ishikawa and HEC-1B cells. *P< 0.05.
Fig 2: Expressions of SPARC in in human endometrial tissues and analysis of the public databases. SPARC expressions of (A) proliferative stage of normal human endometrium, (B) secretory phase of normal human endometrium, (C and D) well-differentiated endometrial carcinoma, (E and F) poorly differentiated endometrial carcinoma were measured by IHC. (Magnification×200). (G) In TCGA Endometrium Statistics of Oncomine, SPARC copy number in normal endometrium (25) was 1.019 times higher than that in endometrial endometrioid adenocarcinoma (291), and 1.056 times higher than that in endometrial serous adenocarcinoma (50) (P<0.05).
Fig 3: Effects of SPARC knockdown on the EMT-related genes and MMPs. (A) By Western blotting (cropped blot), EMT markers, including E-cadherin, N-cadherin, vimentin, Snail, Slug and Twist, and MMPs (MMP2, MMP9, MMP3 and MMP13) were measured in SPARC shRNA transfected, negative control shRNA transfected and non-transfected Ishikawa and HEC-1B cells. SPARC knockdown significantly decreased the expression of E-cadherin, and increased the expression of N-cadherin, Vimentin, Snail, Slug, Twist, MMP3, MMP2 and MMP9, at protein levels. And there was no significant difference in MMP13 and TGF-ß expression between SPARC shRNA-transfected group and negative control group. (B) By qRT-PCR, we confirmed that SPARC down-regulation repressed the expression of E-cadherin, and enhanced the expression of N-cadherin, Vimentin, Snail, Slug, Twist, MMP3, MMP2 and MMP9, at mRNA levels. And three was also no significant change in MMP13 and TGF-ß expression between SPARC shRNA-transfected group and negative control group. The process of EMT was significantly promoted by SPARC down-regulation. *P < 0.05.
Fig 4: SPARC interference increases the expression of gap junction-associated genes associated in HRCECs treated with glucose. (A) Expression of Cx37, (B) Cx40 and (C) Cx43. HRCECs were incubated with glucose for 24 h. Gene expression was examined using reverse transcription-quantitative polymerase chain reaction. Data were presented as mean ± standard deviation (n=3). **P<0.01 and ***P<0.001 vs. negative control group. SPARC, secreted protein acidic and rich in cysteine; HRCECs, human retinal capillary endothelial cells; Cx, connexin.
Fig 5: SPARC interference increases the expression of genes associated with adherens junctions in HRCECs treated with glucose. (A) Expression of VE-cadherin, (B) CTNNA1, (C) CTNNB1 and (D) CTNND1. HRCECs were incubated with glucose for 24 h. Data were presented as mean ± standard deviation (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. negative control group. SPARC, secreted protein acidic and rich in cysteine; HRCECs, human retinal capillary endothelial cells; VE, vascular endothelial; CTNNA1, catenin alpha 1; CTNNB1; catenin beta 1 CTNND1, catenin delta 1.
Supplier Page from Abcam for Anti-SPARC antibody [SP205]