Additional file 1 of The malignancy suppression and ferroptosis facilitation of BCL6 in gastric cancer mediated by FZD7 repression are strengthened by RNF180/RhoC pathway
Creators: name: Guo, Shiwei
name: Deng, Jingyu
name: Wang, Pengliang
name: Kou, Fan
name: Wu, Zizhen
name: Zhang, Nannan
name: Zhao, Zhenzhen
name: Nie, Yongzhan
name: Yang, Lili
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: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Fig S1. The expression and the inhibition to proliferation of BCL6 on GC cells lines. A The protein expression levels of BCL6 in GC cells and immortalized normal gastric epithelial cell (GES-1) were examined by western blotting assay. B Expression levels of BCL6 in AGS and SGC-7901 cells infected with two BCL6 knockdown virus and empty virus were analyzed by western blotting assay (**p&lt;0.01,***p&lt;0.001). C BCL6 inhibits the colony formation of AGS and SGC-7901 cells (**p&lt;0.01, ***p&lt;0.001). Fig S2. Knockdown of BCL6 promotes malignant phenotypes in AGS and SGC-7901 cells. A, B, C Depletion of BCL6 promoted the viability and proliferation of AGS and SGC-7901 cells (Scale bar, 100μm) (***p&lt;0.001). D Depletion of BCL6 promoted the migration of AGS and SGC-7901 cells (Scale bar, 200μm) (***p&lt;0.001). E Depletion of BCL6 promoted the migration and invasion of AGS and SGC-7901 cells (Scale bar, 100μm) (**p&lt;0.01, ***p&lt;0.001). F Depletion of BCL6 promoted the EMT process of AGS and SGC-7901 cells. Fig S3. BCL6 inhibits the growth of GC cell. Tumor growth curves and tumor weight showed the inhibitory effect of BCL6 on tumor in vivo (N=8) (***p&lt;0.001). Fig S4. BCL6 inhibits Wnt/β-catenin pathway by supressing FZD7. A β-catenin protein levels of GC cells after BCL6 knockdown combined with Wnt inhibitor IWP2 treatment (5 µM, 12h). B Representative immunohistochemistry of FZD7 from GC tissue microarrays (Original magnification, ×100 and ×400). C Representative immunohistochemistry of FZD7 and β-catenin in tumor xenografts derived from SGC-7901-NC, SGC-7901-BCL6 cell lines (Original magnification, ×400). D, E ChIP analysis of BCL6 binding to the FZD7 promoter in the 293T cells. The matched IgG was used as a negative control and vicinity region (VR) (-901bp―0 bp) also as a control (***p&lt;0.001). F Western blot for nuclear and cytoplasmic β-catenin in AGS and SGC-7901 cells transfected with control vector (Ctrl1+Ctrl2, Ctrl1+FZD7, BCL6+Ctrl2) or co-transfected BCL6 plasmid and FZD7 plasmid (BCL6+FZD7). Fig S5. TP63 mediates the regulation of GPX4 by the BCL6/FZD7 pathway. A FZD7, TP63 and GPX4 mRNA levels in AGS and SGC-7901 cells transfected with control vector (Ctrl+shCtrl, Ctrl+shTP63, FZD7+shCtrl) or co-transfected FZD7 plasmid and shRNA targeting TP63 (FZD7+shTP63) (*p&lt;0.05, **p&lt;0.01, ***p&lt;0.001). B BCL6, FZD7, TP63 and GPX4 mRNA levels in AGS and SGC-7901 cells transfected with control vector (Ctrl1+Ctrl2, Ctrl1+TP63, BCL6+Ctrl2) or co-transfected BCL6 plasmid and TP63 plasmid (BCL6+TP63) (*p&lt;0.05, **p&lt;0.01, ***p&lt;0.001). Fig S6. IHC staining analysis of RNF180, RhoC and BCL6 in GC tissue microarrays. A Representative immunohistochemistry of RNF180 from GC tissue microarrays (Original magnification, ×100 and ×400). B Representative immunohistochemistry of RhoC from GC tissue microarrays (Original magnification, ×100 and ×400). C Correlations between the the expression of BCL6 and RhoC according to the IHC of TMAs. BCL6 negtively correlated with RhoC in human GC tissues (N=131). Fig S7. RhoC represses BCL6 expression in GC by reducing H3K27ac enrichment on its promoter through HDAC1. A Effects of RhoC on HDAC1, H3K9ac and H3K27ac in GC cells. B, C The HDAC1 inhibitor Pyroxamide (10μM, 24h) restores RhoC repression of H3K27ac and BCL6 (***p&lt;0.001). D knockdown of RhoC significantly reduced the enrichment of HDAC1 on the promoter region of BCL6 (**p&lt;0.01, ***p&lt;0.001).E RhoC reduced H3K27ac enrichment on the BCL6 promoter region (**p&lt;0.01, ***p&lt;0.001). Fig S8. RNF180 promotes the expression of BCL6 in nucleus through RhoC and strengthened its inhibitory effect on downstream genes. A The upstream or downstream proteins expression of BCL6 in AGS and SGC-7901 cells transfected with control vector (Ctrl1+Ctrl2, Ctrl1+RhoC, RNF180+Ctrl2) or co-transfected RNF180 plasmid and RhoC plasmid (RNF180+RhoC). B Western blot for nuclear and cytoplasmic BCL6 in AGS and SGC-7901 cells transfected with control vector (Ctrl1+Ctrl2, Ctrl1+RhoC, RNF180+Ctrl2) or co-transfected RNF180 plasmid and RhoC plasmid RNF180+RhoC). Fig S9. Expression of BCL6 and its downstream genes is regulated by RNF180 and RhoC. A RNF180, RhoC, BCL6, FZD7, TP63, GPX4 mRNA expression in AGS and SGC-7901 cells transduced with control vector (Ctrl+Lenti-siCtrl, Ctrl+Lenti-siBCL6, RNF180+Lenti-siCtrl) or co-transduced RNF180 plasmid and Lenti-si BCL6 virus (RNF180+Lenti-siBCL6) (**p&lt;0.01, ***p&lt;0.001). B RhoC, BCL6, FZD7, TP63, GPX4 mRNA expression in AGS and SGC-7901 cells transduced with control vector (shCtrl+Lenti-siCtrl, shCtrl+Lenti-siBCL6, shRhoC+Lenti-siCtrl) or co-transduced shRhoC plasmid and Lenti-si BCL6 virus (shRhoC+ Lenti-siBCL6) (**p&lt;0.01, ***p&lt;0.001). Fig S10. Expression of BCL6 and its downstream proteins is regulated by RNF180 and RhoC. A RNF180, RhoC, HDAC1, H3K27ac, BCL6, FZD7, TP63, GPX4 protein expression in AGS and SGC-7901 cells transduced with control vector (Ctrl+Lenti-siCtrl, Ctrl+Lenti-siBCL6, RNF180+Lenti-siCtrl) or co-transduced RNF180 plasmid and Lenti-si BCL6 virus (RNF180+Lenti-siBCL6). B RhoC, HDAC1, H3K27ac, BCL6, FZD7, TP63, GPX4 protein expression in AGS and SGC-7901 cells transduced with control vector (shCtrl+Lenti-siCtrl, shCtrl+Lenti-siBCL6, shRhoC+Lenti-siCtrl) or co-transduced shRhoC plasmid and Lenti-si BCL6 virus (shRhoC+ Lenti-siBCL6). Fig S11. Malignancy reppression and ferroptosis facilitation of BCL6 in GC cells are up-regulated by RNF180. A, B BCL6 depletion reversed the inhibition of cell proliferation, migration and invasion induced by RNF180 overexpression in AGS and SGC-7901 cells(Scale bar, 100μm) (**p&lt;0.01,***p&lt;0.001). C, D BCL6 depletion reversed cell death induced by erastin or RSL3 in RNF180-overexpressing AGS and SGC-7901 cells (***p&lt;0.001). Table S1. Correlation of BCL6 expression to clinicopathological features in gastric cancer patients. Table S2. The Primer Sequences Used in this Study.