Supplementary Data from Targeting Prostate Cancer Subtype 1 by Forkhead Box M1 Pathway Inhibition
Authors: Ketola Kirsi , Munuganti Ravi S.N. , Davies Alastair , Nip Ka Mun , Bishop Jennifer L. , Zoubeidi Amina 
Published: April 2023

<p>Supplementary Figure S1. GSEA analyses of subtype-specific genes (PCS1, PCS2 and PCS3) in 42DENZR and 42FENZR cells. Supplementary Figure S2. A) Expression of FOXM1 in different cell lines. Total proteins were extracted from LNCaP, 16DCRPC and ENZR 42D and 42F cells and western blot was performed using FOXM1 antibody (Abcam, 1:1000). Vinculin (1:5000) was used as a loading control. FOXM1 band densitometry was normalized to vinculin. B) Upstream regulator analysis of FOXM1 in PCS1 signature and 42DENZR and 42FENZR cells assessed by IPA (red: target gene overexpression, green: target gene downregulation, orange arrow: target gene is known as induced and blue arrow: target gene known as reduced by pathway activation). C) Expression of FOXM1 pathway expression in metastatic prostate cancer patients analysed using Genesapiens database (20). Supplementary Figure S3. A) Effect of Mon and Thiostrepton effect on FOXM1 transcriptional activity, 42DENZR cells were treated with 100 nM of Mon and Thiostrepton for 24 hours and FOXM1 luciferase activity was performed. B) Monensin reduces FOXM1 protein level. Monensin effect (0, 1, 10, 50 and 100 nM) on FOXM1 protein was addressed using Western blot in 42DENZR cells. Total protein were extracted and western blot was performed using FOXM1 antibody (1:1000). Vinculin (1:5000) was used as a loading control. C) Effect of Mon on cell cycle. Flow cytometry was performed to analyse cell cycle population after Mon treatment for 0 to 72h, percentage of sub-G1 cells in response to Mon in 16DCRPC, 42DENZR and 42FENZR cells was evaluated. D) DARTS assay in presence and absence of Mon in pronase treated samples indicated Mon binding to FOXM1. GAPDH was used as a control. E) Analysis of the effect of Mon on FOXM1 pathway by IPA Upstream regulator analysis of Mon exposed 42DENZR cells (green: target gene downregulation, blue arrow: target gene known as reduced by pathway inactivation). Supplementary Figure S4. Portion of high CD49b (CD49b+/CD24-) cells in 16DENZR, 42DENZR and 42FENZR cells. B) Effect of Mon and C) FOXM1 silencing by FOXM1 siRNA on portion of CD49b+/CD24- cells. Supplementary Figure S5. The effect of FOXM1 inhibition on ALDH activity. A) Portion of ALDH active cells in 16DENZR, 42DENZR and 42FENZR cells. B) The effect of Mon and C) FOXM1 silencing on ALDH activity in 42DENZR and 42FENZR cells. D) Cell proliferation of ALDHHigh and ALDHLow cells (separated from 42DENZR and 42FENZR cells using FACS and Aldefluor ALDH assay) in response to Mon measured by CTG cell proliferation assay. E) FOXM1 protein expression in 2 ALDHHigh and ALDHLow 42DENZR and 42FENZR cells assessed by western blot of FOXM1 and vinculin (loading control). Supplementary Figure S6. A) Mouse body weight (g) in response to Vehicle and Monensin (Mo) treatments for 3.5 weeks. B) Effect of Mon on ALDH activity in 42DENZR tumor xenografts in vivo assessed by Aldefluor ALDH assay and FACS. Supplementary Figure S7. A) The expression of PCS1 signature is enriched in CRPCNeuroendocrine patients. Fold changes of genes in PCS1 signature in CRPC-NE compared to CRPC-Adeno prostate cancer patients are presented as a heatmap. B) The expression of PCS1 signature is enriched in Trp53 and Pten (NPp53 mice) that fail to respond to abiraterone and differentiate to neuroendocrine prostate cancer. Fold changes of genes in PCS1 signature in NPp53 abiraterone-"exceptional non-responders" compared to NPp53 vehicle are presented as a Heatmap. C) PCS1 signature in wild type (WT), single (SKO, (PBCre4:Pten f/f :Rb1 f/+ )), double (DKO, PBCre4:Pten f/f :Rb1 f/f ) and triple knock out (TKO, PBCre4:Pten f/f :Rb1 f/f :Trp53 f/f ) mice . Fold changes of genes in PCS1 signature in SKO, DKO and TKO mice compared to WT mice are presented as a heatmap.</p>