Additional file 1 of Hypoxia increases RCC stem cell phenotype via altering the androgen receptor (AR)-lncTCFL5-2-YBX1-SOX2 signaling axis
Creators: name: Guo, Changcheng
name: Sun, Yin
name: Zhai, Wei
name: Yao, Xudong
name: Gong, Dongkui
name: You, Bosen
name: Huang, Chi-Ping
name: Zheng, Junhua
name: Chang, Chawnshang
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: 19999 Mathematical Sciences not elsewhere classified
subject: FOS: Mathematics
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Computational Biology

description: Additional file 1: Figure S1. The expression of protein level related to AR. (A) The AR protein level expression under hypoxia (H) and normoxia (N) in OSRC-2 and SW839 cells. (B) The EZH2 and SRC expression under hypoxia (H) and normoxia (N) in OSRC-2 cells. (C) The efficiency of shRNA-AR at the protein level (left) in SW839 cells and the efficiency of oe-AR in OSRC-2 cells (right) determined at mRNA level. (D) The HIF1α expression under hypoxia (H) and normoxia (N) in OSRC-2 cells. Figure S2. The supplement figures demonstrated how to focus on lncTCFL5-2. (A) The fold change of lncRNAs in microarray analysis of SW839 cell inresponse to hypoxia. (B) The list of the top 20 downregulated lncRNAs by hypoxia. (C) SW839 and OSRC-2 cells were lentivirally transduced with sh-AR and oe-AR, respectively, and then cells were exposed to hypoxia or normoxia for 2 days. Total RNAs were analyzed by Q-PCR for the 20 down regulated lncRNAs. (D) OSRC-2 cells were virally transduced with oe-AR and pWPI, and then cells exposed to hypoxia (H) or normoxia (N) for 2 days. Q-PCR was used to show 3 lncRNAs expressions. The lncRNA expressions were calculated by hypoxia/normoxia. (E) SW839 and OSRC-2 cells were lentivirally transduced with sh-lncTCFL5-2 sequence 1 and sh-lncTCFL5-2 sequence 2, then cells were exposed to hypoxia or normoxia for 2 days. Sphere formation assay was used to demonstrate the CSCs number. (F) RCC cells were lentivirally transduced with sh-lncTCFL5-2 or oe-lncTCFL5-2 then exposed to hypoxia and normoxia for 2 days. AR expression was evaluated by qPCR and Western-blot. Figure S3. The supplement figures demonstrated AR/lncTCFL5-2/YBX1/SOX2 signaling axis. (A) ACHN cells were lentivirally transduced with oe-AR and cells exposed to hypoxia or normoxia for 2 days, qPCR analysis of the expression of lncTCFL5-2. (B) OSRC-2 cells were treated with the anti-androgen enzalutamide (Enz) (10 μM) for 48h, qPCR was used to test the expression of CSCs biomarkers, including CD24, CD133, PAX2, SOX2 and CD105. (C) The AR and YBX1 protein levels were tested in immunoprecipitates with anti-AR and anti-YBX1 antibodies in 293T cells. (D) The lncTCFL5-2 can be detected in the immunoprecipitate with anti-YBX1 at endogenous level in OSRC-2 cells. (E) OSRC-2 cells were lentivirally transduced with oe-AR or oe-AR oe-mutant lncTCFL5-2 or oe-AR oe-lncTCFL5-2 and cells exposed to hypoxia for 2 days, qPCR analysis of the expressions of AR and lncTCFL5-2. (F) OSRC-2 cells were exposed to hypoxia and normoxia for 2 days. Then immunofluorescent staining was used to detect the CD24 and CD133 double-stained cells. Figure S4. The relationship of AR, YBX1, lncTCFL5-2, and SOX2. (A) The AR, YBX1, and SOX2 expression in ccRCC tumor tissues and normal tissues Based on mining TCGA Data. (B) Real-time RT-PCR assays for detecting the correlation analysis of SOX2 and YBX1 or lncTCFL5-2. (C) SW839 and OSRC-2 cells were lentivirally transduced with sh-HIF2α, or treated with a small molecule inhibitor and then exposed to hypoxia or normoxia for 2 days. Total RNA was analyzed for expression of lncTCFL5-2 by real-time PCR.