Fig 1: In vitro cellular uptake of APODOX-anti-PSMA in prostate cancer cell lines. (a) Expression profiles of GAPDH, TfR, SCARA5 and PSMA and affinity binding of APODOX-anti-PSMA to PSMA on LNCaP and 22RV1 prostate cancer cell lines. The individual blots were cropped from different parts of the same membrane (as indicated by dividing white spaces). Right part shows the densitometric analysis of expression of TfR, SCARA5 and PSMA and binding to PSMA relatively compared to the expression of the house-keeping protein GAPDH. The densitometric analyses were performed on uncropped images using AzureSpot software. The values are expressed as means ± standard deviations of independent triplicates. Vertical bars indicate standard deviation. *Indicate significant differences (p < 0.05) between the two cell lines. (b) DOX fluorescence obtained from continuous quantitative phase imaging of LNCaP cells treated with DOX/APODOX/APODOX-anti-PSMA and the competitive assay between APODOX-anti-PSMA and anti-TfR/anti-PSMA antibodies. (i) DOX fluorescence micrographs obtained at various time points of treatment. (ii) DOX fluorescence values from cells treated with DOX/APODOX/APODOX-anti-PSMA at various time points expressed as means ± standard deviations of six independent measurements. Vertical bars indicate standard deviation. *Indicate significant differences (p < 0.05) among the tested groups. (iii) DOX fluorescence values from competitive assay between APODOX-anti-PSMA and anti-TfR/anti-PSMA antibodies expressed as means ± standard deviations of six independent measurements. Vertical bars indicate standard deviation. *Indicate significant differences (p < 0.05) between cells pre-treated with antibodies and APODOX-anti-PSMA cellular uptake without competition. (c) Stability of mouse anti-PSMA binding to APODOX evaluated by incubation of APODOX-anti-PSMA with 10 mg·mL−1 human IgG for 0, 0.5, 24 and 48 h with subsequent detection of mouse antibodies conjugated on APODOX (i) and released into solution (ii). (d) 12.5% SDS-PAGE (i) and native-PAGE (ii) gels showing protein content of DOX (1) APO (2), APO after disassembly and reassembly (3), APODOX (4) and APODOX-anti-PSMA (5). Marked are the > 250 kDa APO, ~60 kDa antibody, and ~19 kDa light subunit. (e) Size, ζ-potential and TEM micrographs (inset) of APO (i), APODOX (ii) and APODOX-anti-PSMA (iii).
Fig 2: SCARA5 induced G2/M cell cycle arrest by inhibiting FOXM1. (A, B) The inhibitory effect of SCARA5 on cycle related markers was verified by q-PCRand WB. (C) After overexpression of SCARA5, the DNA damage repair marker phosphorylated histone H2AX and checkpoint protein was detected by q-PCR and WB. (D) The common transcription factors of CyclinB1, CDC25C, CHK1, and DEGs of GSE12667 was shown in Venn diagram. (E) The mRNA expression of FOXM1 was downregulated in SCARA5-expressed A549 cells. (F) Ectopic expression of FOXM1 attenuated the inhibitory effect of SCARA5 on CDC25C, Cyclin B1, and CHK1. (G) FOXM1 protein could combine with the promoters of CyclinB1 and CDC25C. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3: SCARA5 promoter was hypermethylated in lung cancer. Methylation status of 64 lung cancer tissue and 25 normal tissue sample was detected by Methylation specific PCR (MSP), 57/64 (89%) primary tumor tissues detected hyper- methylation of promoter, but none of normal lung tissues was detected hypermethylation.
Fig 4: SCARA5 induced cell cycle arrest and apoptosis in lung cancer. (A) The SCARA5 expression of 71 lung cancer samples in GSEA12667 was arranged by log2 median intensity. (B) Gene enrichment plots showed that the gene set G2M_CHECKPOINT was enriched in SCARA5-Low subgroup. (C, D) The effects of SCARA5 on apoptosis in A549 and H1299 cells were detected by flow cytometry analysis too. Representative flow cytometry plots and histogram statistics of apoptosis changes. (E, F) The effects of SCARA5 on cell cycle in A549 and H1299 cells were detected by flow cytometry analysis. Representative flow cytometry plots and histogram statistics. Data was presented as mean ± SD, *p < 0.05, ***p < 0.001.
Fig 5: SCARA5 was associated with endoplasmic reticulum function and induced HSP70 family proteins upregulated. (A) Volcano map of DEGs in vector- and SCARA5-expressed A549 cells. (B, C) Go and KEGG analysis showed SCARA5 related to endoplasmic reticulum and unfold protein reaction. (D) DEGs related to UPR were mainly Heat shock protein family proteins. (E, F) Upregulation of HSP70 in SCARA5-expressed A549 cells was confirmed by q-PCR and WB. (G) Confocal microscopy showed subcellular localization of SCARA5 was similar to ER. *p < 0.05, **p < 0.01, ***p < 0.001.
Supplier Page from Abcam for Anti-SCARA5 antibody