Additional file 1 of TSPAN18 facilitates bone metastasis of prostate cancer by protecting STIM1 from TRIM32-mediated ubiquitination
Creators: name: Zhou, Qianghua
name: Chen, Xu
name: Yao, Kai
name: Zhang, Yangjie
name: He, Haixia
name: Huang, Hao
name: Chen, Hao
name: Peng, Shengmeng
name: Huang, Ming
name: Cheng, Liang
name: Zhang, Qiang
name: Xie, Ruihui
name: Li, Kaiwen
name: Lin, Tianxin
name: Huang, Hai
Publisher: name: figshare
Year: 2023
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Supplemental Table 1. The antibodies used in this study are listed as follows. Supplemental Table 2. The primers used in real time qPCR are listed as follows. Supplemental Table 3. Basic characteristics of prostate cancer patients in two cohorts. Supplemental Table 4. Univariate analysis of prognostic factors correlated with OS and CSS. Supplemental Table 5. Multivariate analysis of prognostic factors correlated with OS and CSS. Supplemental Figure 1. (a) Co-IPanalysis of interaction between exogenous STIM1 and exogenous TSPAN18 in HEK-293Tcells, transfected with Flag-TSPAN18 and Myc-STIM1 plasmid using anti-Mycantibody(left) or anti-Flag antibody (right). (b) Co-IP analysis of interaction between Flag-TSPAN18 and Myc-Orai1 using anti-Flag antibody (left) or anti-Mycantibody (right) in HEK-293T cells. (c)Co-IP analysis of interaction between Flag-TSPAN18 and Myc-Orai1 using anti-Flag antibody in Flag-TSPAN18 overexpressing DU145 cells. Supplemental Figure 2. The Western Blot analysis of TSPAN18 and STIM1 protein level in DU145 and PC-3 cells treated as indicated. The values are expressed as the mean ± s.d. of three independent experiments. **p&lt;0.01, ***p &lt; 0.001, ANOVA with post hoctest or Student’s t test. Supplemental Figure 3. The qRT-PCR analysis of TSPAN18 and STIM1 mRNA level in DU145 and PC-3 cells treatedas indicated. The values are expressed as the mean ± s.d.of three independent experiments. **p&lt;0.01, ***p &lt; 0.001,ANOVA with post hoc test or Student’s t test. Supplemental Figure 4. The Western Blot analysis of TSPAN18 and STIM1 protein level in DU145 and PC-3 cells treated asindicated. The values are expressed as the mean ± s.d.of three independent experiments. **p&lt;0.01, ***p &lt; 0.001,ANOVA with post hoc test. Baf: Bafilomycin, ns: no significance. Supplemental Figure 5. The representative peptide of TRIM32(a) or MIB1 (b) from mass spectrometry. Supplemental Figure 6. (a)Co-IP analysis of interaction between exogenous STIM1 and exogenous TRIM32 inHEK-293T cells transfected with His-TRIM32 plasmid and Myc-STIM1 plasmid usinganti-Myc antibody(left) or anti-His antibody (right). (b-c) The Western Blotanalysis of TRIM32 and STIM1 protein level in DU145 and PC-3 cells treated asindicated. The values are expressed as the mean ± s.d. of three independent experiments. ***p &lt; 0.001, Student’s t test or ANOVA with post hoctest. (d) The protein level of STIM1 in DU145 and PC-3cells transfected with scramble or si-TRIM32 were monitored by WB at indicated times after cycloheximide (CHX, 20μg/mL). Supplemental Figure 7. The representative time-lapse images of cytosolic Ca2+level within PC-3 cells transfected with indicated siRNAs or plasmids. Scale bars: red, 50 μm. Supplemental Figure 8. The representative time-lapse images of cytosolic Ca2+level within indicated cells. Scale bars: red, 50 μm. Supplemental Figure 9. (a-b) Cell viability wasevaluated in TSPAN18 knockdown or overexpressing DU145 and PC-3 cells. (c)Tumor growth curves are summarized in the line chart. The average tumor volumeis expressed as the mean ± SD of six mice. (d) Representative images of thetumors of TSPAN18 knockdown or overexpression groups and their respectivecontrols. (e) Tumor weights were measured after the tumors were surgicallydissected. ns:no significance. Supplemental Figure 10. The Western Blot analysis of E-cadherin and N-cadherinprotein level in DU145 and PC-3 cells treated as indicated. The values are expressed as the mean ± s.d. ofthree independent experiments. **p&lt;0.01, ***p &lt; 0.001,ANOVA with post hoc test or Student’s t test. Supplemental Figure 11. The representative images of migration and invasion assays usingTSPAN18-overexpressing or control DU145 and PC-3 cells transfected withindicated siRNAs (a, b) or treated with SKF96365 or DMSO (c, d). Supplemental Figure 12. The Western Blot analysis of N-cadherin and E-cadherinprotein level in DU145 and PC-3 cells treated as indicated. The values are expressed as the mean ± s.d. ofthree independent experiments. **p&lt;0.01, ***p &lt; 0.001,ANOVA with post hoc test. ns: no significance. Supplemental Figure 13. Therepresentative bioluminescence images of the mice after 6 weeks of inoculationswith indicated PC-3 cells. Supplemental Figure 14. The expression difference of TSPAN18 between lowT stage PCa tissues and high T stage PCa tissues in Cohort 1 (a) and Cohort 2 (b).ns: no significance.Student’s t test.