Additional file 1 of STIL/AURKA axis promotes cell proliferation by influencing primary cilia formation in bladder cancer
Creators: name: Li, Jingxian
name: Qi, Yuanjiong
name: Li, Bo
name: Liu, Yan
name: Yang, Kuo
name: Zhang, Zhihong
name: Zhu, Jianqiang
name: Du, E.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Hematology

description: Additional file 1: Figure S1. PC is deficient in human bladder cancer cells, and PC assembly is enhanced under serum-starved conditions. (A) Normal bladder epithelium cells (SV-HUC-1) and bladder cancer cells (BIU87, EJ, T24, and 5637) were serum-starved for 48 h, then fixed and stained with antibodies against ace-tubulin (red) and DAPI (blue) for immunofluorescence analysis. The scale bar indicates 10 μm. (B) The percentage of ciliated cells in SV-HUC-1, BIU87, EJ, T24, and 5637 cells. (C) Control, 24 h serum-starved, 48 h serum-starved, and serum re-stimulation after 48 h serum-starved EJ cells were stained with antibodies against ace-tubulin (red) and DAPI (blue) for immunofluorescence analysis. (D) The percentage of ciliated cells in Control, 24 h serum-starved, 48 h serum-starved, and serum re-stimulation after 48 h serum-starved EJ cells. (E) The expression levels of acetylated tubulin and ARL13B and quantification of Control, 24 h serum-starved, 48 h serum-starved, and serum re-stimulation after 48 h serum-starved EJ cells (n = 3, mean ± SEM). Unpaired t-test analysis was utilized to compare the differences between the two groups. *P-value &lt; 0.05, **P-value &lt; 0.01, ***P-value &lt; 0.001, ****P-value &lt; 0.0001. Figure S2. PC deficiency could promote the cell cycle and proliferation of BLCA cells. (A-C) SV-HUC-1 and EJ cells transfected with siNC and siIFT88 were serum-starved for 48 h, then fixed and stained antibodies against ace-tubulin (red) and DAPI (blue) for immunofluorescence analysis. The percentage of ciliated cells in different groups was quantification. (D) Western blot analysis for the protein expression (IFT88 and acetylated tubulin) in the control and IFT88-knockdown cells. The percentage of protein levels was quantification. (E) Cell cycle analysis of IFT88-knockdown and control BLCA cells. (F) The viability of IFT88-knockdown and control BLCA cells was measured by CCK8 assay. (G, H) Western blot analysis for the protein expression (including IFT88, acetylated tubulin, ARL13B, SHH, SMO, and GLI1) in IFT88-knockdown and control BLCA cells. The percentage of protein levels was quantification. Unpaired t-test analysis was utilized to compare the differences between the two groups. *P-value &lt; 0.05, **P-value &lt; 0.01, ***P-value &lt; 0.001, ****P-value &lt; 0.0001. Figure S3. STIL regulates migration, proliferation, growth, and cycle in T24 cells. (A, B) The Kaplan–Meier survival curves were depicted to show the progression-free survival (PFS) and recurrence-free survival (RFS) in the high/low STIL expression group. (C, D) The migration ability for STIL knockdown/over-expression T24 cells compared to their corresponding control group by the wound healing assays. (E, F) The viability of STIL knockdown/over-expression T24 cells compared to their corresponding control group was measured by CCK8 assay. (G-I) The cell growth for STIL knockdown/over-expression T24 cells compared to their corresponding control group by colony formation assays. The number of colonies was counted utilizing the software image J. (J, K) Cell cycle analysis of STIL knockdown/over-expression T24 cells compared to their corresponding control group. Unpaired t-test analysis was utilized to compare the differences between the two groups. *P-value &lt; 0.05, **P-value &lt; 0.01, ***P-value &lt; 0.001, ****P-value &lt; 0.0001. Figure S4. STIL locates at the centrosome and regulates PC formation. (A) The EJ cell was serum-starved for 48 h, then fixed and stained antibodies against γ-tubulin (red), STIL (green), and DAPI (blue) for immunofluorescence analysis. (B) The expression levels of STIL and quantification of Control, 24 h serum-starved, 48 h serum-starved, and serum re-stimulation after 48 h serum-starved EJ cells (n = 3, mean ± SEM). Unpaired t-test analysis was utilized to compare the differences between the two groups. (C) No serum-starved and 48 h serum-starved EJ cells were stained antibodies against acetylated tubulin (red), STIL (green), and DAPI (blue) for immunofluorescence analysis. (D-E) The STIL knockdown or overexpression and their corresponding control T24 cells were serum-starved for 48 h, then fixed and stained antibodies against ace-tubulin (red) and DAPI (blue) for immunofluorescence analysis. The percentage of ciliated cells were compared between the control and treatment group. (F) Western blot analysis for the protein expression (including STIL, IFT88, acetylated tubulin, ARL13B, SMO, and GLI1) in STIL-knockdown and control T24 cells. The percentage of protein levels was quantification. (G) Western blot analysis for the protein expression (including STIL, IFT88, acetylated tubulin, ARL13B, SHH, SMO, and GLI1) in STIL-overexpression and control T24 cells. The percentage of protein levels was quantification. (H) Control and STIL over-expressed T24 cells were fixed and stained with antibodies against GLI1 (red) for immunofluorescence analysis. The scale bar indicates 10 μm. (I) siNC and siSTIL T24 cells transfected with siIFT88 or not, respectively, were serum-starved for 48 h, then fixed and stained with antibodies against ace-tubulin (red) and DAPI (blue) for immunofluorescence analysis. The scale bar indicates 10 μm. The percentage of ciliated cells was quantification. (J and K) The protein expression levels (including STIL, acetylated tubulin, ARL13B, IFT88, SHH, and GLI1) in siNC and siSTIL T24 cells transfected with siIFT88 or not were respectively detected by western blot analysis. All the percentages of protein levels were quantification. Unpaired t-test analysis was utilized to compare the differences between the two groups. *P-value &lt; 0.05, **P-value &lt; 0.01, ***P-value &lt; 0.001, ****P-value &lt; 0.0001. Figure S5. STIL regulated AURKA in T24 cells. (A, B) The Kaplan–Meier survival curves were depicted to show the progression-free survival (PFS) and recurrence-free survival (RFS) in high/low AURKA expression. (C, D) The western blot analysis for AURKA protein expression in STIL depletion/overexpression T24 cells and their corresponding normal control. (E, F) The western blot analysis for the protein expression (STIL, AURKA, acetylated tubulin, ARL13B, and IFT88) in AURKA depletion/overexpression T24 cells and their corresponding normal control. (G) The protein expression levels (STIL, AURKA, acetylated tubulin, ARL13B, IFT88, SHH, GLI1, and SMO) in siNC and siSTIL T24 cells transfected with siNC or siAURKA respectively were detected by western blot analysis. (H) The protein expression levels (STIL, AURKA, acetylated tubulin, ARL13B, IFT88, SHH, GLI1, and SMO) in control and STIL overexpressed T24 cells transfected with siNC or siAURKA respectively were detected by western blot analysis. All the percentages of protein levels were quantification. Unpaired t-test analysis was utilized to compare the differences between the two groups. *P-value &lt; 0.05, **P-value &lt; 0.01, ***P-value &lt; 0.001, ****P-value &lt; 0.0001. Figure S6. STIL influences cell migration, proliferation, growth, and PC formation by regulating AURKA. (A, B) siNC and siSTIL T24 cells transfected with siNC or siAURKA, respectively, were serum-starved for 48 h, then fixed and stained with antibodies against ace-tubulin (red) and DAPI (blue) for immunofluorescence analysis. The scale bar indicates 10 μm. The percentage of ciliated cells was quantification. (C, D) Control and STIL overexpressed T24 cells transfected with siNC or siAURKA respectively were serum-starved for 48 h, then fixed and stained with antibodies against ace-tubulin (red) and DAPI (blue) for immunofluorescence analysis. The scale bar indicates 10 μm. The percentage of ciliated cells was quantification. (E) The migration of control and STIL overexpressed T24 cells transfected with siNC or siAURKA were analyzed by wound-healing assay. (F, G) The growth and viability of control and STIL overexpressed T24 cells transfected with siNC or siAURKA were analyzed by CCK8 and colony formation assay. (H, I) The western blot analysis for protein expression (STIL and AURKA) in vector and STIL overexpressed T24 cells treated with different concentrations of cycloheximide (CHX). (J, K) The western blot analysis for protein expression (STIL and AURKA) in siNC and siSTIL T24 cells treated with different concentrations of cycloheximide (CHX). All the percentages of protein levels were quantification. Unpaired t-test analysis was utilized to compare the differences between the two groups. *P-value &lt; 0.05, **P-value &lt; 0.01, ***P-value &lt; 0.001, ****P-value &lt; 0.0001.