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. 2024 Jan 1;14(2):714–737. doi: 10.7150/thno.88057

Figure 1.

Figure 1

ALDH1A1 and ALDH1A3 regulate the CSC phenotype and PCa radiosensitivity. (A) The RNA sequencing analysis of LNCaP cells transfected with ALDH1A1 siRNA, ALDH1A3 siRNA, or scrambled siRNA revealed that ALDH1A3 downregulation is associated with a decrease in CSC-related gene expression at a larger extent than ALDH1A1 (n = 41, RT2 Cancer Stem Cells geneset). Blue arrows indicate genes whose expression levels were confirmed by western blotting in panel H. (B) Gene Set Enrichment Analysis (GSEA) for genes significantly up- or down regulated upon ALDH1A1 or ALDH1A3 knockdown revealed that deregulated genes are associated with stemness and differentiation. (C) Flow cytometry analysis of ALDH+ population upon ALDH1A1 and ALDH1A3 knockdown shows decreased Aldefluor enzymatic activity. N≥3; Error bars = SD; *p < 0.05. (D) Percentage of the spherogenic cells after ALDH1A1 or ALDH1A3 depletion. The bar graph represents the % of spherogenic cells upon ALDH1A1 and ALDH1A3 knockdown. N ≥ 3; Error bars = SD; *p < 0.05. (E) Quantitative real-time PCR (RT-qPCR) analysis of ALDH1A1 expression in LNCaP and DU145 parental and radioresistant cell lines. N = 3; Error bars = SD; *p < 0.05. (F) Relative cell radiosensitivity was analyzed by 2D radiobiological colony forming assay after siRNA-mediated knockdown of ALDH1A1 or ALDH1A3 in LNCaP, C4-2B, or PC3 cells. Cells transfected with scrambled (Scr) siRNA were used as control. N ≥ 3; Error bars = SD; *p < 0.05; **p < 0.01; ***p < 0.001. (G) The RNA sequencing analysis of LNCaP cells transfected with ALDH1A1 siRNA, ALDH1A3 siRNA or scrambled siRNA revealed that ALDH1A1 downregulation is associated with a decrease in DNA damage response (DDR) and repair genes (n = 71, RT2 DNA Damage Signaling Pathway geneset); *p < 0.05. Blue arrows indicate genes whose expression levels were confirmed by western blotting in panel H. (H) Western blot analysis of selected genes from the datasets in Figure 1A and Figure 1G. Representative images of one of four independent repeats are shown; Error bars = SD; *p < 0.05; **p < 0.01. (I) GSEA analysis for genes significantly up- or down regulated upon ALDH1A1 knockdown revealed their association with Chk2 signaling and DNA double strand break repair.