Lyn Tyrosine Kinase Regulates Androgen Receptor Expression and Activity in Castrate Resistant Prostate Cancer

Anousheh Zardan, Ka Mun Nip, Daksh Thaper, Paul Toren, Sepideh Vahid, Eliana Beraldi, Ladan Fazli, Francois Lamoureux, Kilian Gust, Michael E. Cox, Jennifer L.

Bishop and Amina Zoubeidi

Supplementary Figures

Supplementary Figure S1:

CRPCNormal Primary PCa

Src

Supplementary Figure S1: Src expression in the progression of PCa. Src expression was evaluated in human tissue specimens by immunohistochemistry from normal prostate specimens or patients with primary hormone naïve PCa or CRPC (TURP-Transurethral Resection of Prostate).

Supplementary Figure S2: : Lyn overexpression increases AR transcriptional activity: A) Lyn expression in different stably transfected Lyn clones: Proteins were extracted from LNCaPLyn-Clone#A, LNCaPLyn-Clone#B and LNCaPMock cells and western blot was performed using Lyn antibody, vinculin was used as a loading control. B: LNCaPLyn-Clone #A, LNCaPLyn-Clone#B and LNCaPMock cells were transfected with Probasin-luciferase and were treated +/- R1881 for 24h PSA luciferase activity was determined. The results were reported as mean ± SD; *p<0.05 and ***P<0.0001.

Mock

Vinculin

Lyn

Clone#A Clone#B

Lynoverexpression

A

B

Supplementary Figure S2:

Supplementary Figure S3: Lyn knockdown inhibits AR transcriptional activity: A) Lyn expression in different shRNA-Lyn stably transfected clones: Proteins were extracted from LNCaPsh-Lyn#1, LNCaPsh-Lyn#2 and LNCaPsh-Ctr cells and western blot was performed using Lyn antibody, vinculin was used as a loading control. B: from LNCaPsh-Lyn#1, LNCaPsh-Lyn#2 and LNCaPsh-Ctr cells were transfected with Probasin-luciferase and were treated +/- R1881 for 24h PSA luciferase activity was determined. The results were reported as mean ± SD and ***P<0.0001.

 

sh-Ctr sh-Lyn#1 sh-Lyn#2

Vinculin

Lyn

A

B

Supplementary Figure S3:

Supplementary Figure S4:

Lyn

AR

PSA

Vinculin

LNCaP

Ctr Lyn #1Lyn #2

siRNA

Supplementary Figure S4: Lyn knockdown affects AR expression: LNCaP cells were transfected with 10nM of siRNA control, siRNA-Lyn #1 or siRNA-Lyn #2. 48 hours post transfection, total proteins were extracted and western blot was performed Lyn, PSA and AR antibodies and Vinculin was used as a loading control.

Supplementary Figure S5:

Supplemental Figure S5: Src kinase siRNA treatment does not affect AR expression. LNCaP cells were transfected with different concentrations of siRNA control, siRNA-Src. 48 hours post transfection, total proteins were extracted and western blot was performed Src, PSA and AR antibodies and Vinculin was used as a loading control.

Src kinase

AR

PSA

Vinculin

5 10 20 5 10 20 (nM siRNA)

Ctr Src kinase

Supplementary Figure S6:

Supplemental Figure S6: Lyn knockdown does not affect cell growth: LNCaPsh-Lyn and LNCaPsh-Ctr were grown in FBS and cell growth was assessed using crystal violet over time.

LYN Mean STDEV Cores Case SQRT SEBPH 0.8 0.5 70.0 18 5.9 0.1

Untreated cancer 0.9 0.7 152.0 78 4.5 0.2CRPC_TURP 2.0 1.1 20.0 9 4.5 0.2

Table S1: Statistical data from Lyn TMA analysis

Supplementary Table S1:

Src Mean STDEV Cores Case SQRT SEBPH 1.6 0.7 35.0 18 5.9 0.1

Untreated cancer 2.0 0.7 155.0 78 4.9 0.2CRPC_TURP 2.5 0.6 18.0 9 4.2 0.1

Table S2: Statistical data from Src TMA analysis

Supplementary Table S2: