Additional file 3 of CYP1B1-catalyzed 4-OHE2 promotes the castration resistance of prostate cancer stem cells by estrogen receptor α-mediated IL6 activation
Creators: name: Lin, Qimei
name: Cao, Jiasong
name: Du, Xiaoling
name: Yang, Kuo
name: Yang, Xu
name: Liang, Zhixian
name: Shi, Jiandang
name: Zhang, Ju
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 2. Additional results figures. Figures S1-S12 show additional data related to the results shown in the main figures. Figure S1. The plasmid construction process and plasmid maps. A, pcDNA3.1( +)-CYP1B1 plasmid construction diagram. B-D, Plasmid maps of pcDNA3.1( +)-CYP1B1, CRISPRi-CYP1B1 and CRISPRi-Lac. Figure S2. Transfection efficiency/cytotoxicity of the plasmids. pcDNA3.1( +) plasmids, which expresses 3 × flag-tagged peptide, were transfected into LNCaP cells. The transfection efficiency (A) and cytotoxicity (B) of the plasmids were assessed. Scale Bar = 50 μm. Figure S3. Selected ion monitoring (SIM) chromatograms of the 4-OHE2 standard. E2 was dissolved in methanol. The monitored ion in the mass spectrum had a mass/charge ratio of 755 m/z and a retention time of 6.95 min. Figure S4. Validation of CYP1B1 silencing efficiency in LNCaP-abl and PC3 cells and CYP1B1 overexpression efficiency in LNCaP cells by western blotting. Figure S5. HPLC–MS assay results showing the concentration of intracellular 4-OHE2 in CYP1B1 knockdown or control PC3 cells (t-test). *p &lt; 0.05. Figure S6. The effect of CYP1B1 on the expression of stemness-associated genes. A, Characteristics of differential gene expression profiles of CYP1B1High and CYP1B1Low PRAD samples from the TCGA. B, The expression of some basal cell markers and stemness-associated genes was verified by qRT-PCR in CYP1B1-overexpressing LNCaP cells compared with control LNCaP cells (t-test). C, Representative IF staining of CD44 (red) and CYP1B1 (green) in CRPC tissue. Scale bar = 50 μm. D, The expression of CYP1B1 and stemness-associated genes was tested in spheroid cells (PCSCs) and adherent cells (Bulk) by qRT-PCR (t-test). E–F, Relative mRNA levels of some stemness-associated genes were verified by qRT-PCR in PC3 (E) and LNCaP-abl (F) cells after CYP1B1 knockdown (t-test). All values represent the means ± SD from three independent experiments. *p &lt; 0.05. Figure S7. IL6 expression was tested in spheroid cells (PCSCs) and adherent cells (Bulk) by qRT-PCR. All values represent the means ± SD from three independent experiments. *p &lt; 0.05. Figure S8. IF staining of CYP1B1 (red) and IL6 (green) in PC3 cells. Scale bar = 50 μm. Figure S9. IF staining of CD44 (red) and IL6 (green) in PC3 cells. Scale bar = 50 μm. Figure S10. qRT-PCR analysis of the indicated IL6-STAT3 target genes in LNCaP-abl (A) and PC3 (B) cells treated with 4-OHE2 or IL6 (one-way ANOVA). *p &lt; 0.05. Figure S11. qRT-PCR analysis of the indicated IL6-STAT3 target genes in LNCaP-abl and PC3 cells treated with 4-OHE2 and/or AB-IL6 (one-way ANOVA). *p &lt; 0.05. Figure S12. IF staining of ERα (red) and CD44 (green) in PC3 cells.