Fig 1: MRL MSC produced and released higher amount of MANF than BL6, BALB/c and DBA1 MSC. (A) Proteomic analysis of differential expression of MANF, GAS6, NENF, and SEMA5A in MRL MSC compared to BL6 MSC secretomes. Effect size indicate the standardized mean difference in protein expression level between MRL MSC and BL6 MSC. For each protein, the median intensity levels (Log 2 value) in MRL MSC and BL6 MSC are indicated. Normalized protein intensities were used to calculate the Effect size MRL/BL6. (B) Western blot analysis of MANF, GAS6, NENF, and SEMA5A in whole-cell extracts from BL6, BALB/c and DBA1 MSC. The intensity value of each target protein band was normalized against the intensity value of α-Tubulin gel band used as the internal loading control for each sample. (C) MANF mRNA expression level in MRL MSC and BL6 MSC. Western blot analysis of supernatants of BL6, BALB/c, and DBA1 MSC showing blotted proteins stained with Ponceau S (D) and the MANF protein band revealed by the anti-MANF antibody (E). MRL MSC whole cell extract was used as a positive control.
Fig 2: circSEMA5A is upregulated in BC. (A) Diagram showed the circSEMA5A derived from exons 3 and 4 of SEMA5A gene. The back-splicing junction site of circSEMA5A was confirmed by PCR amplification and followed by Sanger sequencing. (B) The relative expression of circSEMA5A was detected by qRT-PCR in BC tissues and paired normal bladder tissues. (C) The relative expression of circSEMA5A was detected by qRT-PCR in BC cell lines and a normal human uroepithelial cell line. (D and E) The transfection efficiencies were verified by qRT-PCR when T24 cells were transfected with circSEMA5A overexpression plasmids and UM-UC-3 cells were transfected with circSEMA5A siRNAs. Data are presented as mean ± SD. *P < 0.05.
Fig 3: CircSEMA5A accelerates BC angiogenesis through upregulating SEMA5A. (A and B) qRT-PCR and western blot assays detecting SEMA5A expressions after T24 cells were transfected with circSEMA5A overexpression plasmids and UM-UC-3 cells were transfected with circSEMA5A siRNAs. (C and D) Angiogenesis capability was assessed by tube formation assay after T24 cells were transfected with circSEMA5A overexpression plasmids together with SEMA5A siRNAs and UM-UC-3 cells were transfected with circSEMA5A siRNAs together with SEMA5A overexpression plasmids. (E) Binding motif of EIF4A3 in circSEMA5A and SEMA5A mRNA. (F and G) RIP validated the interactions of EIF4A3 with circSEMA5A and SEMA5A mRNA. (H and I) Cells were transfected with circSEMA5A or EIF4A3 overexpression plasmids or siRNAs, the enrichments of SEMA5A mRNA in anti-EIF4A3 precipitates were analyzed by qRT-PCR and compared with NC group. (J and K) The influence of circSEMA5A or EIF4A3 on the stability of SEMA5A mRNA was assessed by RNA stability analysis. Data are presented as mean ± SD. NS, no significance; *P < 0.05.
Supplier Page from Abcam for Anti-SEMA5A antibody