Additional file 1 of Flavokawain A is a natural inhibitor of PRMT5 in bladder cancer
Creators: name: Liu, Shuangjie
name: Liu, Zhuonan
name: Piao, Chiyuan
name: Zhang, Zhe
name: Kong, Chuize
name: Yin, Lei
name: Liu, Xi
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Information Systems not elsewhere classified
subject: Cancer
subject: Hematology

description: Additional file 1: Supplementary Methods. Supplementary Table. Correlation between expression of PRMT5 clinicopathological parameters in 60 cases of bladder cancer patients. Fig. S1. (a) PRMT5 expression correlated with BLCA gene signatures in the TCGA dataset. (b) PRMT5 expression correlated with BLCA marker genes in the TCGA dataset. (c) Kaplan–Meier analysis of survival prognosis based on PRMT5 expression in Imvigor datasets. (d) IHC analysis of PRMT5 in different stage BC. (e) Western blot analysis of PRMT5 expression and histone methylation level in BC tissues and adjacent normal bladder tissues. Statistical analysis of protein expression was shown on the right side. Fig. S2. (a) Cell viability assay performed by treating FKA in urothelial cell SV-HUC-1. (b) Cell viability assay performed after treatment with FKB, and FKB IC50 were calculated in T24 (left) and UMUC3 (right). Data are represented as mean ± SD in three replications. (c) Cell viability assay performed after treatment with FKC, and FKC IC50 were calculated in T24 (left) and UMUC3 (right). Data are represented as mean ± SD in three replications. (d) PRMT5 expression changed with different concentrations of FKB treatment times in T24 (upper) and UMUC3 (lower). (e) PRMT5 expression changed with different concentrations of FKC treatment times in T24 (upper) and UMUC3 (lower). (f) Different PRMT expression changed with different concentrations of FKA in T24 (left) and UMUC3 (right). Fig. S3. (a) Cell apoptosis measured by knocking down PRMT5 expression, FKA treatment, and supplied FKA in PRMT5 overexpressed UMUC3 using flow cytometry. (b) Apoptosis rates for replicated assays were counted. (c) Cell viability assay performed after treatment with FKA or PRMT5 shRNA combined with cetuximab in T24 (left) and UMUC3 (right). Data are represented as mean ± SD in five replications. (d) Functional pathway enrichment of predicted FKA downstream targets. (e) Bladder cancer regulon genes changes after supplying FKA in UMUC3 measured by PCR, and the fold changes were standardized and normalized by log10. Fig. S4. (a) Bio-Layer Interferometry detecting the combination and dissociation constants of FKA and human recombinant PRMT5. (b) Fluorescent report CETSA assay confirmed that 25 μM FKA treatment could inhibit PRMT5 degradation when heated, thus enhancing PRMT5 stability in UMUC3. (c) Fluorescent report CETSA assay performed for PRMT5 expression when treated with FKA in wild-type and Y304A/ F580A mutated PRMT5 in UMUC3 cells. (d) Conservation of PRMT5 Y304 and F580 in different species.